Characterization of two Friunavirus phages and their inhibitory effects on biofilms of extremely drug resistant Acinetobacter baumannii in Dakar, Senegal

Abstract

Background: Acinetobacter baumannii is a gram-negative, opportunistic pathogen, that is responsible for a wide variety of infections and is a significant cause of hospital-acquired infections. A. baumannii is listed by the World Health Organization (WHO) as a critical priority pathogen because of its high level of antibiotic resistance and the urgent need for alternative treatment solutions. To address this challenge, bacteriophages have been used to combat bacterial infections for more than a century, and phage research has regained interest in recent years due to antimicrobial resistance (AMR). However, although the vast majority of deaths from the AMR crisis will occur in developing countries in Africa and Asia, few phages' studies have been conducted in these regions. In this study, we present a comprehensive characterization of the bacteriophages vAbBal23 and vAbAbd25, actives against extremely drug-resistant (XDR) A. baumannii.

Methods: Phages were isolated from environmental wastewaters in Dakar, Senegal. The host-range, thermal and pH stabilities, infection kinetics, one step growth assay, antibiofilm activity assay, sequencing, and genomic analysis, were performed to characterize the isolated phages.

Results: Comparative genomic and phylogenetic analyses revealed that vAbBal23 and vAbAbd25 belong to the Caudoviricetes class, Autographiviridae family and Friunavirus genus. Both phages demonstrated activity against strains with capsular type KL230. They were stable over a wide pH range (pH 3 to 9) and at temperatures ranging from 25 °C to 40 °C. Additionally, the phages exhibited notable activity against both planktonic and biofilm cells of targeted extremely drug resistant A. baumannii. The results presented here indicate the lytic nature of vAbBal23 and vAbAbd25. This is further supported by the absence of genes encoding toxins, resistance genes and bacterial virulence factors, highlighting their potential for future phage applications.

Conclusion: Phages vAbBal23 and vAbAbd25 are promising biological agents that can infect A. baumannii, making them suitable candidates for use in phage therapies.

Keywords: Acinetobacter baumannii; Antimicrobial resistance; Bacteriophage; Characterization; vAbAbd25; vAbBal23.

Fig. 1

Plaque morphology of phage vAbBal23 (clear plaque 2 mm with a halo) and vAbAbd25 (clear plaque, < 1 mm without a halo) on isolate AB12

Fig. 2

Stability of phages vAbBal23 and vAbAbd25 under various conditions. (A) and (B) Effects of temperature on the stability of phages. The phage was incubated at different temperatures for 1 h. (C) and (D) Effects of pH on the stability of phages vAbBal23 and vAbAbd25. The phage was incubated for 1 h at different pH values. 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

Fig. 3

In vitro planktonic cell lysis assay evaluating phage A) vAbBal23 against strain AB12, B) vAbAbd25 against strain AB12 and C) a combination of phages vAbBal23-vAbAbd25 against strain AB12. Each phage was studied at multiplicities of infection (MOIs) 1, 10^− 1, and 10^− 3. The data represent the means and standard errors from three biological and technical triplicate experiments for single phage treatment and triplicate experiments for phage cocktail treatment. D) One-step growth curve of phages vAbBal23 and vAbAbd25 on AB12. The data points indicate the PFUs/mL at different time points. Each data point represents the mean of three independent measurements

Fig. 4

Efficacity of phages vAbBal23 and vAbAbd25 to reduce preformed biofilms for strains (A) AB12, (B) AB13 and quantification of planktonic and biofilm (C) AB12, (D) AB13 after phage treatments. The biofilm biomass and bacterial cell viability were determined by the crystal violet assay and the colony counting method, respectively. The data are illustrated in a violin plot derived from a biological triplicate and technical triplicate experiments. The statistical significance is denoted as “*” for p-value < 0.05, “**” for p-value < 0.01, “***” for p-value < 0.001, and “****” for p-value < 0.0001

Fig. 5

Predicted coding sequences (CDSs) of phages (A) vAbBal23 and (B) vAbAbd25 with transfer RNAs (tRNAs), transfer-messenger RNAs (tmRNAs), virulence factors (VFs), antimicrobial resistance genes (AMRs), clustered regularly interspaced short palindromic repeats (CRISPRs) and functional annotation of the CDSs

Fig. 6

Phylogenetic and comparative genomic analyses of the phages vAbBal23 and vAbAbd25. (A) Phylogenetic tree of vAbBal23 and vAbAbd25 generated by VICTOR using the whole-genome sequences of phage homologs in BLASTn. (B) Percentage sequence similarity between phages calculated using VIDIRIC