Complete genome sequence of Pseudomonas aeruginosa YK01, a sequence type 16 isolated from a patient with keratitis

Abstract

Objectives

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is frequently associated with multidrug resistance and global epidemic outbreaks, contributing significantly to morbidity and mortality in hospitalized patients. However, P. aeruginosa belonging to the sequence type (ST) 16 was rarely reported. Here, this report presents the complete genome sequence of YK01, a ST16 P. aeruginosa isolate from a patient with keratitis. The complete reference genome of P. aeruginosa YK01 is expected to provide valuable data for investigating its genomic population, enhancing understanding of genetic basis of P. aeruginosa species complex.

Data description

A complete genome of 6.3 Mb was obtained for P. aeruginosa YK01 by combining Illumina 150-bp short reads and Nanopore long reads. The assembly is fully complete with chromosomal genome size of 6,183,266 bp, presenting a GC content of 66.7%, and a plasmid with the size of 46,067 bp, presenting GC content of 59.0%. Predicted chromosomal genomic features include 5,709 CDS, 12 rRNAs, 63 tRNAs, 4 ncRNAs, and 5,788 genes. To our knowledge, this genome data represents the first complete genome of P. aeruginosa ST16, providing crucial information for further comparative genome analysis.

Objective

P. aeruginosa poses a significant threat to hospitalized patients, contributing to high morbidity and mortality rates [1, 2]. The emergence of multidrug-resistant and extensively drug-resistant P. aeruginosa strains, particularly carbapenem-resistant isolates, has become a critical concern, prompting the World Health Organization (WHO) to elevate P. aeruginosa to high priority on the 2024 Bacterial Priority Pathogens List [3]. P. aeruginosa is a ubiquitous opportunistic pathogen causing diverse infections, ranging from pulmonary infections in cystic fibrosis patients to cutaneous and ocular manifestations [4]. Its role as primary etiological agent in bacterial keratitis, especially among contact lens users [5], is well-established, with studies reporting a worldwide infections with incidence ranging 6.8% to 55% [6]. While genomic analyses have illuminated the characteristics of certain P. aeruginosa lineages, including sequence type (ST) 308 in India [7], ST235 [8], ST1203 in USA [9], the genomic landscape of ST16 remains largely unexplored.

While the PubMLST Pseudomonas aeruginosa multilocus sequence typing (MLST) scheme (accessed 13 September 2024) encompasses 5,007 STs, only six records of ST16 are currently documented. To enhance our understanding of the conservation and evolutionary biology of this rare ST, we present the complete genome sequence of a ST16 P. aeruginosa strain YK01. This study presents the complete genome sequence of a clinically isolated P. aeruginosa strain belonging to ST16, which will contribute to the limited understanding of ST16, a rarely described lineage within the P. aeruginosa species. This study addresses this gap by providing the first comprehensive genomic characterization of a P. aeruginosa ST16 isolate.

Data description

YK01 was isolated from a 51-year-old patient with bacterial keratitis in Shenzhen, China (DataFile 1). Bacterial isolation was performed using sheep blood agar plate. YK01 was initially identified as P. aeruginosa by MALDI-TOF mass spectrometric analysis. To obtain genomic DNA of YK01 for sequencing, a single colony was inoculated in lysogeny broth (LB) at 37 °C with 220-rpm agitation for 20 h. 1 mL of bacterial culture was collected, and the genomic DNA was extracted using the EasyPure® Genomic DNA Kit (TransGen, China). The short-read library was prepared using Nextera XT DNA Library Preparation Kit (Illumina, USA) and sequenced by an Illumina NextSeq 500 platform with 150-bp paired-end reads (Novogene, China). Pair end raw reads (n = 7,728,918; SRA: SRR30916324; DataFile 2) were accessed and trimmed with FastQC v0.12.1 [10] and Trimmomatic v0.39 [11], respectively. The long-read library was prepared with Ligation Sequencing Kit V14 (SQK-LSK114) and sequenced using R10.4.1 flow cell and Nanopore MinION sequencer Mk1B (Oxford Nanopore Technologies, UK). Raw nanopore sequencing data was base-called to generate long reads (SRA: SRR30916323; DataFile 3). A hybrid assembly (DataFile 4 and 5) was performed using Unicycle v0.5.0 [12], followed by genomic functional annotation using the NCBI Prokaryotic Genome Annotation Pipeline [13]. The resulting complete genome of strain YK01 comprising chromosomal sequence and one plasmid sequence, exhibited an average coverage of 346-fold as determined by QUAST v5.2.0 [14]. The completeness of genome was verified using CheckM v1.2.1 [15] (DataFile 6). De novo assembly of strain YK01, analyzed using GTDB-Tk v2.3.2 [16], confirmed the species identification of strain YK01 as P. aeruginosa, closely related to strain NCTC10332 (RefSeq: GCF_001457615.1) with an average nucleotide identity of 99.36% (DataFile 7).

YK01 harbors a 6,183,266 bp chromosome (DataFile 8) and a 46,067 bp plasmid, designated pYK01 (DataFile 9). The genome encodes 239 virulence factors, 27 biocide and metal resistance genes, and five antibiotic resistance genes, all located on the chromosome (DataFile 10). Comparative analysis with PAO1 (GenBank: AE004091.2) [17] and PA14 (GenBank: NC_008463.1) [18] revealed the absence of wzy and wzz genes in YK01, along with partial deletions within the pil and pvd gene clusters (DataFile 11). The genes associated with biofilm formation in YK01 are same to those in PAO1 and PA14, primarily encompassing the alginate synthesis genes (alg gene cluster) and quorum sensing genes (las and rhl gene clusters) (DataFile 11). Alignment of pYK01 with the closely related plasmid pP9Me2 (GenBank: CP118640) indicated the absence of acr genes, which encode proteins that inhibit the Type I-F CRISPR-Cas system [19] (DataFile 12). CRISPRCasFinder [20] identified five CRISPR arrays and a Type I-F CAS system within YK01 (DataFile 13). Furthermore, PHASTEST [21] identified a prophage closely related to Pseudomonas phage YMC11/02/R656 (RefSeq: NC_028657.1). AntiSMASH [22] analysis revealed 16 secondary metabolite biosynthetic gene clusters (BGCs) with potential links to virulence factor expression (DataFile 13 and 8).

To establish the global context of strain YK01, a core-genome single nucleotide polymorphism (SNP) analysis was conducted. This analysis utilized 42 P. aeruginosa public genomes with associated metadata, retrieved from the NCBI database (DataFile 14), and employed Snippy v4.6.0 with the reference genome of strain PAO1 as a reference (DataFile 15) [23]. A maximum-likelihood phylogenetic tree was constructed using IQ-TREE v2.2.0.3 (DataFile 16), employing the GTR + I + G model and 1,000 ultrafast bootstrap replicates [24]. This tree, along with metadata including source, location, and virulence factors, was rooted at sequence GCA 034812525.1 and visualized using iTOL v 6.9.1 (DataFile 17) [25]. All scripts in this study were written into DataFile 18 Table 1.

Table 1 Overview of data files/data sets

Limitations

While this study presents the first complete genome sequence of P. aeruginosa ST16, the analyses are limited by the current scale of available complete genomic data for this specific ST. To overcome this limitation and provide a broader understanding of this lineage, future research should include a larger collection of complete ST16 genomes, potentially leveraging hybrid assembly approaches. Analysis of a broader dataset would facilitate a more robust investigation into the evolution of antibiotic resistance genes carried on plasmids and the comparative genomics of pathogenicity islands within the ST16 population.