# Isolation and characterization of Acinetobacter phage vAbaIN10 active against carbapenem-resistant Acinetobacter baumannii (CRAB) isolates from healthcare-associated infections in Dakar, Senegal

Issa Ndiaye, Laurent Debarbieux, Ousmane Sow, Bissoume Sambe Ba, Moussa Moïse Diagne, Abdoulaye Cissé, Cheikh Fall, Yakhya Dieye, Ndongo Dia, Guillaume Constantin de Magny, et al.

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Issa Ndiaye, Laurent Debarbieux, Ousmane Sow, Bissoume Sambe Ba, Moussa Moïse Diagne, et al. Isolation and characterization of Acinetobacter phage vAbaIN10 active against carbapenem-resistant Acinetobacter baumannii (CRAB) isolates from healthcare-associated infections in Dakar, Senegal. Journal of Global Antimicrobial Resistance, 2025, 41, pp.151-158. 10.1016/j.jgar.2024.12.024. hal-04943292

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# Isolation and characterization of *Acinetobacter* phage vAbaIN10 active against carbapenem-resistant *Acinetobacter baumannii* (CRAB) isolates from healthcare-associated infections in Dakar, Senegal

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Issa Ndiaye<sup>a,g,*</sup>, Laurent Debarbieux<sup>b</sup>, Ousmane Sow<sup>a</sup>, Bissoume Sambe Ba<sup>c</sup>, Moussa Moise Diagne<sup>d</sup>, Abdoulaye Cissé<sup>a</sup>, Cheikh Fall<sup>a</sup>, Yakhya Dieye<sup>a</sup>, Ndongo Dia<sup>d</sup>, Guillaume Constantin de Magny<sup>e,f</sup>, Abdoulaye Seck<sup>a,g</sup>

## ARTICLE INFO

**Article history:**

Received 4 November 2024

Revised 10 December 2024

Accepted 16 December 2024

Available online 30 December 2024

Editor: Professor A Tsakris

**Keywords:**

Bacteriophage

ABRC, vAbalN10

Characterization

Acinetobacter baumannii

AMR

## ABSTRACT

**Background:** Carbapenem-resistant *Acinetobacter baumannii* (CRAB) is a critical antimicrobial resistance threat and a WHO-prioritized pathogen. With intrinsic resistance to multiple antibiotics and the emergence of pan-resistant isolates, CRAB infections are challenging to treat, often relying on polymyxins, tigecycline, aminoglycosides, or combinations, though co-resistance is rising globally. Phage therapy is considered as a potential treatment for multidrug-resistant *A. baumannii*. This study focused on isolating and characterizing phages active against CRAB strains from healthcare-associated infections in Dakar, Senegal.

**Methods:** A lytic phage, *Acinetobacter vAbalN10*, was isolated from wastewater collected at the Aristide Le Dantec Hospital in Dakar, Senegal. Isolation, host range, efficiency of plating, temperature and pH stability, lysis kinetics, one-step growth test, sequencing, and genomic analysis were performed.

**Results:** Phage vAbaIN10 belongs to the class *Caudoviricetes* and the genus *Friunavirus*. Its genome is 40,279 bp in size. Phage vAbaIN10 is stable across a wide pH range (3–9) and temperature range (25°C–60°C). The phage's lytic activity was evaluated at different multiplicities of infection (MOI): MOI 10, 1, and 10⁻¹. All MOIs significantly reduced the growth of host bacteria. The one-step growth curve showed that vAbaIN10 had a latency period of 25 min and a burst size of approximately 4.78 × 10³ phages per infected bacterial cell. No tRNA, mtRNA, clustered regularly interspaced short palindromic repeat, virulence factors, or antibiotic resistance genes were found in the genome.

**Conclusions:** The biological and genomic characteristics of vAbaIN10 meet the requirements for its potential use in phage therapy.

© 2024 The Authors. Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

## 1. Introduction

*Acinetobacter baumannii* is a gram-negative bacterium, nonfermentive, nonmotile, catalase-positive that thrives in diverse environments including soil, water, and sewage, and in healthcare settings. Healthcare-associated Infections (HAIs) caused by *A. baumannii*, ranging from bacteraemia to urinary tract infections, pose sig-

<footnote><sup>a</sup> Pôle de Microbiologie, Institut Pasteur de Dakar, Dakar, Sénégal</footnote>

<footnote><sup>b</sup> Département de Microbiologie, Laboratoire Bactériophage, Bactérie, Hôte, Institut Pasteur, Paris, France</footnote>

<footnote><sup>c</sup> World Health Organization WCARO, Dakar, Senegal</footnote>

<footnote><sup>d</sup> Département de Virologie, Institut Pasteur de Dakar, Dakar, Sénégal</footnote>

<footnote><sup>e</sup> MIVEGEC, Université Montpellier, CNRS, IRD, Montpellier, France</footnote>

<footnote><sup>f</sup> MEEDiN, Montpellier Ecology and Evolution of Disease Network, Montpellier, France</footnote>

<footnote><sup>g</sup> Faculté de Médecine, Pharmacie et Odontostomatologie, Université Cheikh Anta Diop, Dakar, Sénégal</footnote>

<footnote>* Corresponding author. Mailing address: Pôle de Microbiologie, Institut Pasteur Dakar, 36, Avenue Pasteur, Dakar, BP 220, Sénégal.
E-mail address: seydina.indiaye14@gmail.com (I. Ndiaye).</footnote>

<footnote>* Corresponding author. Mailing address: Pôle de Microbiologie, Institut Pasteur Dakar, 36, Avenue Pasteur, Dakar, BP 220, Sénégal.
E-mail address: seydina.indiaye14@gmail.com (I. Ndiaye).</footnote>
nificant challenges, especially for critically ill or immunocompro-
mised patients [1]. Recently attention has focused on *A. bauman-
nii* due to escalating outbreaks and its robust resistance to antibi-
otics and other antibacterial treatments [2]. This has led to inten-
sified efforts to understand its virulence, transmission dynamics,
environmental persistence, and potential therapeutic interventions
[3]. *A. baumannii* is an ESKAPE-E bacterial pathogen (*Enterococcus
faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *A. baumannii*,
*Pseudomonas aeruginosa*, *Escherichia coli*) and has a higher preva-
lence of multidrug-resistant (MDR) strains than other pathogens
do, which contributes significantly to its clinical problems [4].

The emergence and spread of carbapenem-resistant *A. bauman-
nii* (CRAB) have garnered considerable attention, as carbapenems
are among the most efficacious antimicrobial agents against *A. baue-
mannii* infections. CRAB pose significant problems due to its re-
silience to treatment and are associated with high mortality rates
from 8% to 40% [5,6]. The designation of CRAB as a critical pri-
ority pathogen by the World Health Organization (WHO) under-
scores the urgent need for novel antibiotics and alternative treat-
ment modalities [7].

Phage therapy (PT), which utilizes bacteriophages (phages),
viruses that infect bacteria, represents a promising strategy to
combat MDR *A. baumannii* infections. Although they were discov-
ered more than a century ago, phages have exhibited efficacy in
the treatment of various bacterial infections. As an alternative or
complement to antibiotics, phage therapy holds significant poten-
tial in clinical settings [8,9]. In recent years, there have been nu-
merous reports on the isolation and characterization of diverse *A.
baumannii* phages [10–12]. In vivo studies have shown the poten-
tial of phage therapy in managing MDR *A. baumannii* infections
such as pneumonia [5,13,14], wound infections [15,16], and sepsis
[17]. Furthermore, several impactful case studies involving human
subjects [18–20] have demonstrated the potential efficacy of phage
therapy in addressing *A. baumannii* infections. However, limited re-
search in Africa, despite the significant prevalence of *A. baumannii*
observed in low and middle-income countries, highlights the need
to develop phages research in this region to address antimicrobial
resistance (AMR).

In this study, we present the isolation and characterization of
a novel *Acinetobacter* phage vAbaIN10, which targets CRAB strains
isolated from HAIs in Senegal.

**2. Materials and methods**

2.1. Bacterial isolates, antibiotic susceptibility testing and growth conditions

Eleven (11) carbapenem-resistant *A. baumannii* strains from HAIs were obtained between April 2018 and July 2021 from two biobanks of routine labs at two tertiary hospitals in Dakar. The samples sources were pus (n = 4), vaginal swabs (n = 1), bronchial fluid (n = 3), or urine (n = 3). Antimicrobial susceptibility tests of the isolates were performed following the CA-SFM/EUCAST guidelines (version 2023), and the results are presented in Sup. Table S1. Bacteria were cultured in Luria Bertani (LB) broth or agar (Difco Laboratories, Detroit, MI, USA) at 37°C. K locus types (KLs) were obtained by whole genome sequencing followed by bioinformatic analysis using Kaptive 2.0 [21]. Bacterial growth was monitored turbidimetrically by measuring the optical density at 600 nm (OD<sub>600</sub>), with an OD unit of 1.0 corresponding to 3 × 10<sup>8</sup> cells/mL.

## 2.2. Phage isolation and purification

Wastewater samples were centrifuged (5000 g, 10 min at 4°C),
and the supernatants were filtered through a 0.22-µm-pore-size
membrane (Millex, Syringe Filter). Phage presence was assessed

using the double-layer agar method on the bacterial strains. In
this process, 10 µL of the sample was added onto a freshly pre-
formed lawn of the bacterial cell on soft LB agar (0.7%) layered
over double-concentrated LB agar (1.4%). A sterile pipette tip was
used to select a single plaque, which was then solubilized in phos-
phate buffer saline solution. Four microliters (4 µL) of the serial di-
lution of the phage were then spotted onto an agar plate to purify
the plaques. This procedure was repeated three times. The purified
phages were stored at both -4°C and -80°C in 20% (v/v) glycerol
until further experimentation.

2.3. Host range analysis and efficiency of plating (EOP)

The host range of the phages was tested using a spot test on the 11 CRAB isolates. The sequence type, KLs, and O locus were determined via WGS (Sup. Table S2). Briefly, the spot test method in which a bacterial lawn was spread on the top agar, and 4 µL of serially diluted phage lysate were spotted onto the bacterial lawn. After an 18 h incubation, the plates were observed for lysed plaques.

Bacterial strains lysed by *Acinetobacter* phage vAbaIN10 were selected for EOP analysis. The ratio of plaques formed on the test strain to those formed on the host strain lawn was reported as the EOP. The experiments were performed independently in triplicate, with each assay conducted in triplicate.

2.4. Phage thermal and pH stability assay

To assess the thermal stability of phage vAbaIN10, 100 µL of phage (1.38 × 10¹⁰ PFU/mL) was incubated for 1 h at 25°C, 40°C, 55°C, 60°C, 65°C, or 70°C, after which 50 µL aliquots were taken to measure the phage titre. After 10-fold dilution, 4 µL of each serially diluted sample was spotted onto LB agar with the host bacterium (AB6) to determine the phage titre. For the pH stability assay, aliquots were collected after 1 h of incubation in SM buffer with various pH values ranging from 3.0 to 12.0. All experiments were performed independently in triplicate, with each assay conducted in triplicate.

2.5. Lytic profile of bacteria after phage infection

An overnight bacterial culture (200 µL) was diluted in 10 mL of fresh LB and incubated at 37°C until the OD<sub>600</sub> reached 0.2. Bacterial suspensions and phages at different multiplicities of infection (MOIs) (10, 1, and 10<sup>-1</sup>) were added to microplates and incubated at 37°C with agitation. As a control sample, CRAB isolates were incubated with LB without phage. Bacterial growth was monitored by measuring the OD<sub>600</sub> every 10 min for 16 h with agitation using a spectrophotometer. The experiments were independently conducted in triplicate, with each assay conducted in triplicate.

2.6. One-step growth assay

One-step growth experiment were conducted as previously de-
scribed [22]. Briefly, exponential phase *A. baumannii* cultures were
adjusted to 10⁸ CFU/mL and mixed with phage lysate at an MOI
of 10⁻². The phages were allowed to absorb onto the bacterial
surfaces at room temperature for 5 min. The mixture was subse-
quently centrifuged at 10,000 g for 30 s, and the supernatant was
removed. The pelleted cells were resuspended in 20 mL of pre-
heated (37°C) LB broth and centrifugated at 10,000g for 30 s (re-
peated three times). The final resuspended pellet was incubated
at 37°C. Samples were taken at 5 min intervals and the soft agar
overlay method was used to determine the phage titre. The time
between phage infection and progeny release was defined as the
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Plaque morphology of phage vAbalN10 on isolate AB4. Clear medium-sized plaques (3–4 mm) with a halo.</figcaption></figure>

latent period. The ratio of the phage titre to the number of infected bacterial cells was used to calculate the burst size. The experiments were repeated three times.

## 2.7. Phage DNA extraction and whole genome sequencing

Genomic DNA was isolated from high-titre phage stocks (>10¹⁰ PFU/mL). Briefly, 1 mL of phage lysate was treated with 10 µL (20 U) of DNase I and 4 µL of RNase A (20 mg/mL) and incubated for 30 min at 37°C. The mixture was then treated with 20% SDS and 10 mg/mL of proteinase K, followed by an additional 30 min incubation at 37°C. DNA extraction was performed using the phenol-chloroform method and ethanol precipitation [23]. Nuclear acid concentrations were determined using the Qubit 2.0 fluorometer (Thermo Fisher Scientific). Libraries were prepared from 1 ng of DNA using Nextera XT DNA library preparation kits (Illumina, San Diego, CA, USA) following the manufacturer's protocol. WGS was carried out on an Illumina iSeq 100 sequencers utilizing the 300-cycle i1 Reagent V2 Kit (Illumina, San Diego, CA, USA).

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Stability of phage vAbalN10 under various conditions. (A) Effect of pH on the stability of phage vAbalN10. (B) Effect of temperature on the stability of phage vAbalN10. The experiments were independently performed in triplicate with triplicate assay. The data are presented as the means ± standard errors from three replicates. 'ns' means no significant difference, ‘*’ means *P* < 0.05, ‘**’ means *P* < 0.01, ‘***’ means *P* < 0.001, ‘****’ means *P* < 0.0001.</figcaption></figure>

## 2.8. Bioinformatic analysis

Quality control of the reads was performed using FastQC v0.12.1 [24], followed by adaptor trimming with trim-galore v0.6.10 [25]. The trimmed paired-end reads underwent de novo assembly using SPAdes v3.15.5 [26] in careful mode. The assembled contigs were previsualized using Bandage v 0.9.0 [27]. Coverage per contig and assembly validation were performed using BBMap v 35.85 with default parameters [28], and the host contaminating DNA was manually removed. The input genome was sorted and indexed using Samtools v1.18 [29] followed by assembly error correction via Pilon v1.24 with default parameters [30]. The assembled whole-genome sequences were compared with those of other phages for sequence similarity using the Basic Local Alignment Search Tool (BLASTN) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch (accessed on 09 August 2024)). In accordance with ICTV recommendations, the phage taxonomy was refined using taxMyphage (https://ptax.ku.dk/, accessed on 17 August 2024) predicted coding sequences (CDSs), transfer RNAs, transfer-messenger RNAs, virulence factors, AMRs genes, clustered regularly inter-spaced short palindromic repeats, and functional annotations for CDSs were performed using Pharokka v1.3.0 with default parameters [31]. Option ‘pharokka Plotter’ was used for circular genome visualization.

# 3. Results

## 3.1. Phage isolation and purification

Acinetobacter phage vAbalN10 was isolated from hospital wastewater using AB6 as the host bacterial strain. Through phage purification, concentration, and titration methods, a pure, high-titre stock of phage vAbalN10 (10¹⁰ PFUs/mL) was successfully obtained. The plaques produced by phage vAbalN10 are medium-sized (3–4 mm), clear, and surrounded by a halo on a lawn of three CRAB isolates (AB4, AB6, AB7) (Fig. 1).

## 3.2. Host range activity determination and EOP assay

The host range activity of phage vAbalN10 was determined via a spot test and EOP analyses. The spot test results revealed that
<table><caption>**Table 1**
Host range infection and EOP of phage vAbalN10.</caption><thead><tr><th rowspan="2">Strain</th><th rowspan="2">Resistance</th><th rowspan="2">Origin</th><th rowspan="2">K locus</th><th colspan="2">Phage vAbalN10</th></tr><tr><th>Lytic activity</th><th>EOP</th></tr></thead><tbody><tr><td>Acinetobacter baumannii AB1</td><td>XDR</td><td>Pus</td><td>KL3</td><td>-</td><td>-</td></tr><tr><td>A. baumannii AB2</td><td>XDR</td><td>Vaginal swab</td><td>KL17</td><td>-</td><td>-</td></tr><tr><td>A. baumannii AB3</td><td>XDR</td><td>Bronchial fluid</td><td>KL4</td><td>-</td><td>-</td></tr><tr><td>A. baumannii AB4</td><td>XDR</td><td>Urine</td><td>KL47</td><td>+</td><td>0.91</td></tr><tr><td>A. baumannii AB5</td><td>XDR</td><td>Urine</td><td>KL18</td><td>-</td><td>-</td></tr><tr><td>A. baumannii AB6</td><td>XDR</td><td>Bronchial fluid</td><td>KL47</td><td>+</td><td>(Host = 1)</td></tr><tr><td>A. baumannii AB7</td><td>XDR</td><td>Urine</td><td>KL47</td><td>+</td><td>0.45</td></tr><tr><td>A. baumannii AB8</td><td>XDR</td><td>Pus</td><td>KL17</td><td>-</td><td>-</td></tr><tr><td>A. baumannii AB9</td><td>XDR</td><td>Bronchial fluid</td><td>KL152</td><td>-</td><td>-</td></tr><tr><td>A. baumannii AB10</td><td>XDR</td><td>Pus</td><td>KL17</td><td>-</td><td>-</td></tr><tr><td>A. baumannii AB11</td><td>XDR</td><td>Pus</td><td>KL17</td><td>-</td><td>-</td></tr></tbody></table>

+ indicates the production of a lytic zone, - indicates the absence of a lytic zone. The experiments were independently performed in triplicate with triplicate assay. The resistance profile was determined according to Magiorakos et al. [53] with phenotypes provided in Sup. Table S1.

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** *In vitro* planktonic cell lysis assay of phage vAbalN10 against: (A) AB4, (B) AB6, and (C) AB7 at different MOIs. A 1 h timeline was used instead of a 10 min timeline for convenience. The experiments were independently performed in triplicate with triplicate assay.</figcaption></figure>

*Acinetobacter* phage vAbalN10 produced lytic zones against 3 of the 11 CRAB isolates tested. The 3 sensitives isolates (AB4, AB6, and AB7) which shared the same KL47 and were further assessed by EOP analysis. The results revealed that phage vAbalN10 resulted in less productive infection in the CRAB isolates AB4 and AB6 than in the CRAB AB7 isolate (Table 1).

### 3.3. Thermal and pH stability studies

The stability of phage vAbalN10 under various thermal and pH conditions was tested (Fig. 2). The thermal stability was evaluated from 25°C to 70°C for 1 hour. The results showed no significant reduction in phage stability after incubation at 25°C (*P*-value = 0.1601), 40°C (*P*-value = 0.6374), or 55°C (*P*-value = 0.9996) (Fig. 2A). A significant reduction in phage activity was observed at 60°C and 65°C (*P*-value <0.0001) and no viable phage were detected after incubation at 70°C. To assess pH sensitivity, the phage was exposed to pH values ranging from 3 to 9 for

1 hour. There was no significant change in phage viability at pH values of 4, 5, or 9 (*P*-value = 0.4137). The stability of the phage was significantly reduced at pH 12 (*P*-value = 0.0044) (Fig. 2B).

### 3.4. Lysis kinetics

The assessment of a phage's capacity to suppress host strain growth holds pivotal importance for therapeutic applications. Therefore, we investigated the inhibitory potential of phage vAbalN10 against three strains (AB4, AB6, and AB7) (Fig. 3). Phage infection was initiated at various MOIs (10, 1, and 0.1). The results demonstrated a continuous increase in growth for uninfected CRAB isolates, whereas treatment with phage vAbalN10 notably reduced bacterial growth (Fig. 3). Moreover, compared with AB4, vAbalN10 exhibited better infection efficiency against AB6 and AB7 compare to AB4 with 47.23%, 55.52% and 34.33% of reductions, respectively after 16 h of incubation. Notably, no significant differences were observed among the treatments with different MOIs for AB4 and
One Step Growth

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** One-step growth curve of phage vAbalN10 on AB6. Phages were grown in an exponential phase culture of AB6. The data points indicate the PFUs/mL at different time points. Each data point represents the mean of three independent measurements.</figcaption></figure>

AB6. The detection of bacterial resistance to phage vAbalN10 occurred at 3 h post-incubation for AB4 and AB6 and at 4 h for AB7. Complete inhibition of bacterial growth was subsequently observed at 8–9 h across all the conditions. Notably, even an MOI as low as 0.1 effectively inhibited bacterial growth.

3.5. *One-step growth assay*

To identify the different phases of the phage infection pro-
cess, a one-step growth assay of phage vAbalN10 on AB6 was per-
formed. The latent period (the time between initial infection and
the first burst of new phages) was 10 min. After an exponential

rise, vAbIN10 reached a plateau after 25 min, with a burst size of
approximately 4.78 10³ phages/infected bacterial cell (Fig. 4).

3.6. *Genomic analysis*

The whole genome of phage vAbalN10 was comprehensively characterized. Phylogenetic analysis identified phage vAbalN10 as a member of the family Autographiviridae, subfamily Beijerinckvirinae, and genus Friunavirus. The genome size is 40,279 bp with a GC content of 39.27% and the CDS density is 95.83%. A total of 58 predicted genes encoding 58 proteins were identified (Fig. 5).

The functional annotation revealed 32 hypothetical proteins and 26 predicted proteins with known putative functions, which were classified into six functional groups. Nine predicted proteins were associated with phage-DNA, RNA, and nucleotide metabolism, including essential enzymes such as RNA polymerase, endonuclease VII, nucleotidyltransferase, exonuclease, DNA polymerase, ATP-dependent DNA ligase, DNA-binding protein, DnaB-like replicative helicase, and DNA primase.

Proteins relevant to the phage head and packaging functions include internal virion proteins with endolysin domains, internal virion lysozyme motifs, internal virion proteins, major head proteins, head scaffolding proteins, and terminase large and small subunits. Tail-related proteins included tail spike proteins, three tail proteins, and a head-tail adaptor acting as a connector. Host lysis-related proteins include endolysin and holin/anti-holin. Additionally, a membrane protein was implicated in moron, auxiliary metabolic genes, and host takeover processes. No transfer RNAs, transfer-messenger RNAs, clustered regularly interspaced short palindromic repeats, virulence factors, or antibiotic resistance genes were found in the genome (Fig. 5). A detailed description of the CDSs can be found in Sup. Tables S3 and S4.

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Predicted CDSs of phage vAbalN10. Transfer RNAs (tRNAs), transfer-messenger RNAs (tmRNAs), virulence factors (VFs), antimicrobial resistance genes (AMRs), clustered regularly interspaced short palindromic repeats (CRISPRs), and functional annotations of CDSs.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Percentage sequence similarity between closely related phages calculated using taxMyPhage.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Clinker gene cluster comparison of the whole genome of phage vAbalN10 against its closest relative vB_AbaP_APK37 phage, with per cent amino acid identity represented by grayscale links between genomes. Each similarity group is assigned a unique colour.</figcaption></figure>

For comparative genomic analysis, the genome of phage vAbalN10 was aligned using BlastN against related phages selected on the basis of the sequence coverage range (>80%). ANI analysis of closely related phages revealed that phage vAbalN10 represents a new species in the genus *Friunavirus* (ANI <95%) and the name 'Friunavirus snin' was proposed (Fig. 6).

The phage vAbalN10 presented the highest degree of DNA-level similarity with *Acinetobacter* phage vB_AbaP_APK37 (MK257723.1), with 95.23% identity. The visualization of the phages genome alignments through Clinker (Fig. 7), highlighted gene similarities between the two phages. Interestingly, over half of the CDSs in both phages encode proteins of unknown function, while differ-

ences in known CDSs were observed. The phage vAbalN10 harbours a tail spike protein and a nucleotidyltransferase involved in tail structure and DNA/RNA metabolism, respectively. Conversely, phage vB_AbaP_APK37 contains an HNH endonuclease and a DNA-binding protein.

## 4. Discussion

A. baumannii, an opportunistic pathogen, has become increasingly important in both community and hospital-acquired infections and is related to various types of infections. It is recognized as a major MDR pathogen on a global scale and has been listed
by the WHO as one of the critical superbugs requiring the urgent
development of new antibiotics [7]. Given the pressing need for al-
ternative strategies to treat CRAB infections, phage therapy is cur-
rently been re-evaluated, with a few successful compassionate-use
cases reported thus far. In this study, we present the characteriza-
tion of a novel *Acinetobacter* phage, vAbalN10, isolated from hospi-
tal wastewater in Dakar.

Through spot tests and EOP analysis, Acinetobacter phage vAbalN10 demonstrated a narrow host range, specifically targeting KL47 as previously described [32,33]. The capsular polysaccharide (CPS) surrounding A. baumannii cells serves as a primary receptor for phages and is crucial in initiating phage-host interactions [34]. The CPS is a high molecular weight polymer consisting of recurring units of oligosaccharides (referred to as K units) connected by a Wzy polymerase [35,36]. The ability of a phage to infect different bacterial hosts at the species or genus level can vary due to the presence of capsule depolymerases [37]. Some phages, such as those effective against Klebsiella spp., can infect bacteria from various Enterobacterales species [38,39]. In contrast, phages targeting A. baumannii are relatively specific Acinetobacter spp. [40]. A limited host range can be advantageous, as it helps preserve other bacterial species and minimize disruption of the microbiota [41]. Unlike antibiotics, the narrow host specificity of phages could reduce the development of resistance among bacteria [41]. In this study, phage vAbalN10 inhibited the growth of 3 out of the 11 strains (AB4, AB6, and AB7). However, its limited host range could pose a challenge in finding specific phages matching the bacteria's KL for each infection.

Environmental factors significantly affect phage stability and therapeutic efficacy [42]. The phage vAbalN10 displayed notable thermal stability from 25°C to 55°C, with decreased viability at 60°C-65°C and complete inactivation at 70°C. Similarly, it exhibited stability within a pH range of 3–9, with a significant reduction observed at pH 12. These characteristics are consistent with previous findings on A. baumannii phages [12,32,43], suggesting their potential applicability in stable storage and as therapeutic agents against CRAB infections.

The phage vAbalN10 has a small latent period (10 min) and a large burst size (4.78 10³ phages/infected bacterial cell). Studies have reported that phages with long latent periods can be inefficient, while those with short latency may replicate more rapidly and produce more progeny phages [44]. Other A. baumannii phages, such as Abp1, 350 phages/cell [45] TAC1, 454 phages/cell [46], AB1, 409 phages/cell [47], and vB_AbaM_IME285, 450 phages/cell [48], have been documented to have lower burst sizes compared to phage vAbalN10 and suggesting that this phage could be a promising candidate for therapeutic use.

Previous reports have shown that the genome size of *Autographiviridae* phages ranges from 39.425 pb to 47.868 pb [12,43,49,50], similar to that of vAbalN10. Other phages from the *Friunavirus* genus, have demonstrated efficacy in human phage therapy trials [51,52]. Among the 58 predicted genes in phage vAbalN10 genome, 44.83% were identified as having known functions, while more than half of the genes (55.17%) remained hypothetical proteins. Notably, the vAbalN10 genome lacked genes associated with a temperate life cycle (e.g., integrases), transposable elements, antibiotic resistance, or bacterial virulence, supporting its potential suitability for phage therapy. These findings underscore the potential use of phage vAbalN10 examined in this study for therapeutic purposes. Nevertheless, its limited ability to target only a few hosts could restrict its broader therapeutic applications.

**5. Conclusions**

AMR poses a growing threat to public health worldwide, with
the WHO projecting 10 million annual deaths due to AMR by 2050,

90% of which are expected to occur in Africa and Asia. Address-
ing AMR in Africa require a multifaceted approach, encompassing
improved antibiotic stewardship, surveillance systems, healthcare
infrastructure, and the integration of alternative therapies such
as phage therapy. Therefore, studies investigating the potential of
phage therapy as an alternative or complement to antibiotics are
needed. Here we isolated and characterized a lytic phage vAbalN10
which in term of its characteristics represents a potential candidate
for phage therapy against MDR CRAB isolates in Senegal. There-
fore, further studies are needed to validate its putative therapeutic
value.

**Author contributions**

Conceptualization, I.N, G.C.M., and A.S.; methodology, I.N. and M.M.D; software, I.N.; validation, G.C.M., and A.S.; formal analysis, I.N.; investigation, I.N.; resources, O.S., A.C, B.S.B., C.F., Y.D., N.D.; data curation, I.N.; writing - original draft preparation, I.N.; writing - review and editing, L.D., M.M.D., O.S., A.C, B.S.B., C.F., Y.D., N.D., G.C.M. and A.S.; visualization, I.N.; supervision, L.D., G.C.M., and A.S.; project administration, G.C.M., and A.S.; funding acquisition, G.C.M.

**Data availability statement**

The genome assemblies and annotations were deposited in
the European Nucleotide Archive (ENA) under project number PR-
JEB72144 and SRA and assembly accession number ERR12535799.

**Acknowledgements**

The authors thanks Maïmouna Mbanne Diouf for technical as-
sistance for the Sequencing and all the members of the Pole of Mi-
crobiology, Pasteur Institute of Dakar for their precious assistance.

**Funding:** This study was supported by GCDM at the Institut de Recherche pour le Développement (IRD), France.

**Declaration of competing interests:** The authors declare that they have no competing interests.

**Ethical approval and consent to participate:** Not applicable.

**Supplementary materials**

Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jgar.2024.12.024.

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