Complete genome sequences of Providencia bacteriophages PibeRecoleta, Stilesk and PatoteraRojo

Abstract

Objectives

Providencia is a genus of gram-negative bacteria within the order Enterobacterales, closely related to Proteus and Morganella. While ubiquitous in the environment, some species of Providencia, such as P. rettgeri and P. stuartii, are considered emerging nosocomial pathogens and have been implicated in urinary tract infection, gastrointestinal illness, and travelers’ diarrhea. Given their intrinsic resistance to many commonly used antibiotics, this study aimed to isolate and sequence bacteriophages targeting a clinical P. rettgeri isolate.

Data description

Here we report the complete genome sequence of three novel Providencia phages, PibeRecoleta, Stilesk and PatoteraRojo, which were isolated against a clinical P. rettgeri strain sourced from a patient in a metropolitan hospital in Victoria, Australia. The three phages contain dsDNA genomes between 60.7 and 60.9 kb in size and are predicted to encode between 72 and 73 proteins. These three new phages, which share high genomic similarity to two other Providencia phages previously isolated on P. stuartii, serve as important resources in our understanding about Providencia bacteriophages and the potential for future phage-based biotherapies.

Objective

Providencia are a genus of gram-negative bacteria of the family Morganellaceae, closely related to Proteus and Morganella. There are nine currently recognized species within the genus, with P. rettgeri, P. stuartii and P. alcalifaciens the most encountered in the context of human disease. They are commonly associated with nosocomial urinary tract infection [1], particularly in patients with long-term urinary catheters leading to “purple bag syndrome” [2], associated with wounds in burn patients [3], and linked to diarrhea and gastroenteritis in children and travelers [4, 5]. Members of Providencia are intrinsically resistant to commonly used antibiotics including colistin, the last resort antibiotic for multi-resistant gram-negative bacteria [6]. Consequently, Providencia spp., along with other pathogenic bacteria of the order Enterobacterales including Klebsiella pneumoniae, Escherichia coli, Enterobacter spp., Serratia spp., Proteus spp. and Morganella spp. are classified as Critical Priority 1 on the World Health Organizations global priority pathogens list requiring further research and development of new antibiotics [7].

Bacteriophages (phages), viruses that are capable of infecting and killing bacteria, represent a potential solution to this emerging problem. Bacteriophages are ubiquitous in both natural and artificial environments and predicted to be the most abundant biological entities on the planet [8]. Only 26 Providencia phage genomes have thus far been deposited in the NCBI GenBank as of July 2023. Here we have isolated three novel Providencia phages, named PibeRecoleta, Stilesk and PatoteraRojo, by screening worm farm effluent samples obtained from various locations in Victoria, Australia on a clinical isolate of P. rettgeri (strain 9744). Visible phage plaques (approximately 0.3 mm) were picked, subjected to a total of three rounds of purification to ensure each plaque resulted from a single virion, and propagated as previously described [9].

Data description

DNA were extracted from 1 ml phage filtrates (> 10¹⁰ PFU ml^− 1) using a zinc chloride phenol:chloroform-based extraction [9, 10]. Isolated DNA (100 ng) were then prepared for sequencing using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB) followed by sequencing on an Illumina MiSeq using a v3 600-cycle kit (Illumina) to generate 300 bp paired-end reads (n = 491,519–968,088 paired reads). Raw data were filtered using Trim Galore v0.6.4 with default settings (Q scores of ≥ 20, with automatic adapter detection) [11], and assembled with SPAdes v3.9.0 with default settings [12]. The assembled genome of PibeRecoleta was 60,727 bp with a GC content of 49.3% (1248-fold read coverage), Stilesk was 60,924 bp with a GC content of 49.5% (2310-fold read coverage) and PatoteraRojo was 60,728 bp with a GC content of 49.4% (1410-fold read coverage).

Genome termini were identified to be 11-bp 5’ cos overhangs (5’-GTGCGGAGAGC-3’) on all three phages using PhageTerm v1.0.12 [13] and confirmed by manual inspection of raw reads [10]. Genes were identified using Glimmer3 [14] followed by manual adjustment. Genomes were annotated using a combination of searching against the NCBI Conserved Domain Database [15] and the Virfam Webserver [16]. No tRNA genes were detected using tRNAscan-SE v2.0 [17] or Aragorn v1.2.41 [18]. All software was used with default parameters. A total of 72–73 predicted coding sequences were identified in each of the phage genomes. Those of which could be assigned a function (~ 30%) were characteristically organized in functional modules involved in virion morphogenesis and lysis, and DNA replication and nucleotide metabolism (Data file 1, Data file 2) [19].

The three highly syntenic phages described here share high pairwise DNA sequence similarity (78.7-89.7%) as determined using VIRIDIC (Data file 3) [19, 20]. Examination of public databases for sequenced phage genomes similar to these three phages revealed two highly related Providencia phages, Redjac [21] and PSTCR9, which were both isolated on P. stuartii. (Data file 3) [19]. Redjac and PSTCR9 phages, which represent the only two members of the Redjacvirus genus, share over 70% intergenomic similarity to the three Providencia phages sequenced here, indicating PibeRecoleta, Stilesk and PatoteraRojo phages belong to the Redjacvirus genus (Data file 3). Members of the Redjacvirus are also known to share moderate nucleotide similarity (46–47%) and similar genomic organization to phages from the well-studied flagellotropic Chivirus genus (such as Enterobacteria phage Chi) which target members of the Enterobacteriaceae (Data file 3) [19, 20, 22]. Insights into the molecular mechanisms utilized by these Providencia phages may potentially be gained from work already performed on Chivirus phages.

Table 1 Overview of data files/data sets

Limitations

Providencia phages PibeRecoleta, Stilesk and PatoteraRojo represent complete phage genomes. Our understanding of phages targeting Providencia is currently limited by the small sample size of phages isolated and described to date (n = 26). Isolation and description of additional Providencia phages in the future will facilitate increased knowledge of Providencia phage biology and their potential application as biotherapeutics.