Antibiotic-resistant bacteria, such as Carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa, are a growing problem due to factors such as overuse and misuse of antibiotics, gene transfer, and inadequate diagnostics [17]. Gram-negative bacteria are particularly difficult to treat, and infections caused by multidrug-resistant bacteria can lead to increased healthcare costs, morbidity, and mortality. Phage therapy, which uses viruses that attack and replicate inside bacterial cells, is a potential substitute for antibiotics due to its high specificity and ability to target harmful microorganisms without harming human cells [18]. In a previous study by Gallet et al., 2022 and Adams et al., 2008 they conducted a whole genome analysis of Carbapenem-Resistant Klebsiella pneumoniae genomes and multidrug resistant Acinetobacter baumannii, respectively. They found that these strains are causing serious problems in human heath worldwide [19, 20].
In this study, bacteriophage therapy is considerably constructed on phage cocktail. These cocktails can be formulated by combination of various isolates or phages to increase the efficiency to lyse and they also can be re tailored if resistance develops [21]. To lyse the multiple targeted strains of bacteria, instead of one phage, phage cocktails are formulated. There are very specific phages, only infect a single and specific bacterium. A mixture of phages with perfect host ranges called phage cocktail, to increase targeted strains and useful for broader range application can be predicted [22]. In a previous study by Breijyeh et al., 2020 they conducted a study on how antimicrobial resistant occurs and also provide treatment process. They also suggested that instead of using antibiotics, we can provide phage therapy against multi drug resistant bacteria [23].
In current study, multidrug resistant strains of bacteria were selected to start the designing of phage cocktail. The selected strains were Acinetobacter baumannii AB0057, Klebsiella pneumoniae HS11286, Pseudomonas aeruginosa UCBPP-PA1 which are causing serious health problems. Carbapenem resistance was found to be dominant in these bacterial strains. In a previous study by Chen et al., 2019 and Maria et al., 2020 the therapeutic cocktail to combat staphylococcal strain and Vibrio sp. Va-F3 were designed respectively [24, 25].
A total of 11 prophages were detected from three bacterial genomes by Mageeney et al., 2020 including 3 prophages form Acinetobacter baumannii, 7 prophages from Klebsiella pneumoniae and 1 prophage from Pseudomonas aeruginosa. In another study by Mageeney et al., 2020 they predicted the IGEs from 2168 genomes of bacterial strains but they did not perform the designing of phage cocktail for the bacterial strains, which were included in our current study [26]. In a previous study by Bose et al., 2021 they performed the annotation of prokaryotic genomes using the prophage finder program for the prediction of prophages [27].
We used TIGER and Islander algorithms, which were used by Magneeney et al., 2020 for IGE detection including prophage genomes, however, we used these predicted prophages for designing of therapeutic cocktail against the multi drug resistant strains of bacteria. Prophages were identified and characterized to learn more about their possible effects on the biology and pathogenicity of their bacterial hosts. It may be possible to create novel methods for the treatment and prevention of bacterial illnesses by knowing the genetic components which contribute to the pathogenicity of bacteria.
In a previous study by Martinez-Vaz et al., 2020 they used other methods for hunting of phages from bacterial genome. They used PHASTER, a program for detection of phages from bacterial genomes [28]. In another study by Song et al., 2019, Prophage Hunter, an algorithm for hunting of active prophages was used for the detection of prophages [29]. In our study, integrated genomic element-based algorithms were applied, which detected the different islands from bacterial genomes including prophages.
The small prophages having sizes below 11 kb are considered to be non-functional and unable to assemble a mature and viable bacteriophage because an intact phage with complete functional units can disrupt a target bacterium completely [30]. The prophages having size below 30 kb are also considered small and it is very difficult to differentiate between them and other mobile genetic elements [31]. This can pose a challenge when attempting to accurately identify and classify small prophages within prokaryotic genomes, therefore, we have included only those prophages in our study which had a size above 30 kb.
In a previous study by Happel et al., 2022 they found that prophages that were beyond the average size range for bacteriophages in the NCBI Caudovirales database (less than 11.6 kb), were found using five algorithms for identifying phages. These prophages were excluded from further analysis since it was considered improbable that they would perform their function properly. Also, they discarded those phages which have size larger than 800kb, much larger than size of known phage genome size. None of our selected prophages were larger than 60 kb, which suggests that these prophages can code for complete and viable bacteriophages.
In a previous study by Bobay et al., 2014 they performed the probability distributions for genome size of the 68 double stranded DNA temperate caudophages infecting enterobacteria, and discarded those phages which had a size lower than 30 kb, considering them to be non-functional [32].
A total of 472 ORFs were predicted form the 11 prophages. In a previous study by Lavigne et al., 2018 PHIRE algorithm was presented to predict the regulatory elements in bacteriophage genomes [33]. We run GeneMark.hmm program for prediction of open reading frames.
Prokka annotation of these prophage enabled us to acknowledge the features of these prophages including CDs regions, rRNAs (ribosomal RNAs), and tRNAs (transfer RNAs). Mageeney et al., (2020) performed prokka annotation only for P. aeruginosa strains Pae5 and Pae1505, but in current study different strains of bacteria i.e., Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa were used for cocktail designing. In another study about phage cocktail designing by Maria et al., 2020 the auto annotation of phages were performed using Rapid Annotation Using Subsystem Technology. Their work contributed in determination of Podoviridae and Myoviridae bacteriophages against staphylococcal strains of bacteria (34).
This annotation enabled us to learn more about the diversity and development of these mobile genetic elements, as well as their possible interactions with bacterial hosts and other mobile genetic elements, by examining the CDS, rRNA, and tRNA sections of prophages. This knowledge can aid in the creation of fresh tactics for the management and avoidance of bacterial illnesses.
The presence of 3 tRNAs encoded by 11 prophages were discovered through examination of the tRNA regions in the prophage genomes. Given that the host bacteria normally supply the tRNA molecules required for translation [35], this result raises the possibility that these prophages rely on the host translation machinery for protein synthesis. In a past study by Chen et al., tRNAs from vibrio phages were predicted using ARAGORN software. Also, tRNA-Scans-SE was employed for the prediction of tRNAs with their colorful structure [36].
We also detected rRNA genes, which can interact with the host cell's translation and protein production machinery. We did not find any rRNA in predicted prophages, but it does not have any effect on phage functioning because a bacteriophage hijacks host translation machinery and replicates inside the host cell [37].
We used the BLASTP algorithm to annotate the predicted ORFs in the prophage genomes. This allowed us to identify putative functions and potential biological roles of the encoded proteins. The use of the NCBI non-redundant protein database for the BLASTP search ensured that we had access to a comprehensive collection of known protein sequences for comparison. We identified a total of 29, 79, and 85 Open Reading Frames in 3 prophages of Acinetobacter baumannii with maximum identity percentages of 100% and minimum identity percentages of 30.84%, 40.68%, and 42.82% respectively. The Acinetobacter phage 5W was most identical to our query prophages, which is very effective against multidrug-resistant Acinetobacter baumannii.
Chen et al., 2019 performed annotation of the protein sequences of predicted ORFs using BLASTP algorithm. Out of 530 ORFs analyzed, approximately 66.2% (n = 351) demonstrated amino acid sequence identities ranging from 26–100% with sequences present in the NCBI GenBank database [38].
We identified a total of 14, 13, 73, 37, 63, 43, and 22 Open Reading Frames in 7 prophages of Klebsiella pneumoniae with maximum percentage identities of 100% and minimum identity percentages of 93.76%, 27.51%, 87.79%, 93.83%, 59.29%, 70.18%, and 86.84% respectively. BLAST analysis of Klebsiella prophages showed identical results with Klebsiella phage Mulock. We found that Klebsiella phage KPP5665-2 and Klebsiella phage Mulock were used for bacteriophage therapy [39]. In a study conducted by Herridge et al., 2020 many phages including Klebsiella phage Mulock was used against the resistant strains of Klebsiella [36].
A total of 14 ORFs were identified in the prophage of Pseudomonas aeruginosa which showed maximum identity percentage of 100% and minimum identity percentage of 38.22%. We found that Pseudomonas phage Pf1 was most identical, which is effective against other strain of Pseudomonas [40]. We employed the Softberry promoter online analysis tool to identify putative promoter sequences and regions in 11 prophage genomes. Our results revealed that three prophages of Acinetobacter baumannii had 47, 126, and 133 putative promoters, respectively. Among the seven prophages of Klebsiella pneumoniae, the number of putative promoters ranged from 19 to 108. For the single prophage of Pseudomonas aeruginosa, we identified 19 putative promoters. These findings provide insights into the potential regulatory mechanisms of these prophages and their interactions with the host bacteria. Due to their minimum sized genome and presence of virulence factor they were rejected as discussed by Happel et al., 2022.
We also identified Rho-independent transcription terminators in the prophage genomes. The results showed the presence of terminators in various prophages, with 3 prophages of Acinetobacter baumannii containing 18, 53, and 61 terminators, 7 prophages of Klebsiella pneumoniae containing 10, 8, 35, 25, 31, 29, and 9 terminators, and 1 prophage of Pseudomonas aeruginosa containing 2 terminators. The prediction of Rho-independent terminators is essential for understanding the transcriptional regulation of prophage genes and their integration into the host genome [41]. In a previous study by Chen et al., 2020 they predicted terminators for vibrio phages but not perform any analysis for other strains of bacteria which are used in this study.
The Virulence Search program using the Virulence Factor Database (VFDB) was employed to detect virulent genes in the prophage genomes. The analysis revealed that one prophage of Klebsiella pneumoniae harbored Peptidoglycan DD-metalloendopeptidase family protein and undecaprenyl-phosphate glucose phosphotransferase, which are known virulence factors [42]. Additionally, the prophage of Pseudomonas aeruginosa was found to contain Phenazine biosynthesis protein PhzF, which is an isomerase involved in phenazine biosynthesis and has been linked to virulence in P. aeruginosa [43]. Therefore, those prophages which are detected to producing virulent genes are rejected.
In this study, we selected 5 prophages for phage therapy. These phages met all the inclusion criteria. The size of selected prophages was neither less than 30kb nor higher than 800kb. Apart of this the phages which contain virulence factors were also excluded. Dot plot of selected phages was generated, which enabled us to reject K. pneumoniae Prophage (4049881–4085225 as being similar to K. pneumoniae Prophage (1778306–1808606).
Comparative genome analysis of the prophages was conducted using EasyFig and genome maps were created showing the similarities between two identical genomes of prophages. The selected prophages with their highest similar phage sequences were analyzed using the EasyFig 2.2. This allowed us to visualize the similarities and differences between the prophages and their closest homologs. their highest identical prophage 95.67% (ON391949.1) Acinetobacter phage YC#06 which is discussed by Luo et al., 2022 they isolated and characterized and used this virulent phage against multidrug-resistant Acinetobacter baumannii strains, moreover the also made antibiotic mixtures with that phage to enhance the effects of phage therapy. [44]
After performing BLASTn analysis we found that our selected prophage Acinetobacter baumannii 2759376–2809756 was similar with Podoviral Bacteriophage YMC/09/02/B1251 ABA BP (NC_01954.1) by 96.06% identity. This similar bacteriophage belongs to the family Podoviridae and has a double-stranded circular DNA genome with a length of 45,364 bp and a 39.05% GC content. In a study by Jeon et al., 2012 they perform a complete analysis of Bacteriophage YMC/09/02/B1251 ABA-BP and reported that this phage causes the lysis of an isolate of Carbapenem-Resistant Acinetobacter baumannii strain from a septic patient [45].
In a study by Baginska et al., 2023 they purposed a alternative methods of phage therapy instead of using supplement and other antibiotics. They conducted a study on determination of morphology and biological properties of 12 phages including Acinetobacter phage Acba_1, Acinetobacter phage Acba_3, Acinetobacter phage Acba_11, Acinetobacter phage Acba_15 specific against MDR Acinetobacter baumannii. Moreover, after analysis of morphology and biological properties of these phages they were determined to use these phages against MDR Acinetobacter baumannii [46]. These phages are very similar to those phages of Acinetobacter baumannii used in current study.
In a study by Bleriot et al., 2020 genomic analysis of 40 Klebsiella phage which were integrated in 16 strains of Klebsiella pneumoniae which are resistant to carbapenem. They also included those phages which are highest similar to Klebsiella pneumoniae prophages used in current study. They revealed that these phages contain different proteins which were mainly involved in structure of virion, replications, transcription of virion and regulation of lysogenic and lytic cycle [47].
We reviewed available studies on phages having 100% and 99.99% similarity with our prophages, targeting any additional bacterial species or strains in addition to the mentioned strains (Table. 7). The phage (MN688132.1) targeting Klebsiella pneumoniae (1288317–1338719) is reported by Chen et al., 2022, to be capable of targeting Enterobacter hormaechei as well [51]. E. hormaechei is responsible for nosocomial infections, urinary tract diseases and bacteraemia [53]. Bleriot et al., 2020 also reported phages targeting a wide range of Klebsiella strains, among which some of the phages were also included in our study. The phage (MK416019.1) was found to target K. pneumoniae ST11-VIM1 and phage (MK433582.1) was found to target K. pneumoniae ST258-KPC3. In the same way, phages (MN166823.1 and NC_049452.1) were reported for targeting K. pneumoniae ST512-KPC3 and phage (NC_049449.1) was found targeting K. pneumoniae ST437-OXA245 [47][52]. These Klebsiella strains are found to be carbapenemase producing and are majorly responsible for nosocomial infections, pneumonia, and urinary tract infections leading to high morbidity and mortality rates [47]. This investigation suggests that the phage cocktail we have designed, has the potential to target the above-mentioned bacterial strains also, in addition to our selected MDR bacterial strains.
Phylogenetic analysis was conducted on the prophages isolated from Acinetobacter baumannii and Klebsiella pneumoniae, as well as one prophage from Pseudomonas aeruginosa. The sequences chosen for the analysis were selected based on BLASTn, and the neighbour-joining method with 1000 bootstrap replications was used for the analysis using MEGA 11 software. Highest similar sequences prophages were selected and phylogenetic tree was constructed based on large terminase subunits of selected phages. Two critical tasks are carried out by the terminase large subunit during the viral replication cycle. First of all, it acts as an ATP-powered molecular motor to help viral DNA move into empty capsids. It also serves as an endonuclease, cleaving the viral DNA to start and stop the packing activity [48].
The comparative genome analysis and phylogenetic analysis of the isolated phages provide important insights into the diversity and relatedness of the phages. The Klebsiella prophages were found to be present with other known Klebsiella prophages showing close relatedness, whereas the prophages from Acinetobacter baumannii genomes were closely found to be monophyletic. In this study, the highest similar sequences for Acinetobacter baumannii ranged from 99.91–75.38%, indicating a relatively high degree of diversity among the phages. For Klebsiella pneumoniae, a total of 25 highest similar phages were selected with similarities ranging from 99.99–78.95%. This indicates a relatively high diversity within this species, which is consistent with previous studies that have reported a high level of diversity in Vibrio phages and prophages form Pseudomonas strains.
Phage cocktails have the potential to become an important tool in the fight against multidrug-resistant bacteria in the future [49]. One of the main benefits is their specificity, which allows for targeted and personalized treatment. This reduces the risk of disrupting the beneficial bacteria in the body, unlike traditional antibiotics which can lead to collateral damage to the microbiome [50]. Additionally, phages can be isolated and propagated relatively quickly, and can be tailored to target specific strains or combinations of strains. This makes them a promising alternative to antibiotics, especially in cases where conventional antibiotics are ineffective or unavailable. Overall, the use of phage cocktails could revolutionize the treatment of multidrug-resistant bacterial infections and provide new hope for patients with limited treatment options.