Draft genome of a clinical Pseudomonas qingdaonensis isolate from a hospitalized patient at Kenyatta National Hospital, Kenya

The first draft genome of Pseudomonas qingdaonensis, a gram-negative bacteria isolated from hospitalized patients in Kenya, is presented in this study. The genome was assembled using nanopore MinION readings, yielding an assembly of 8,857,650 base pairs made up of 5(five) contigs. The genome sequence of Pseudomonas qingdaonensis will help researchers better grasp its genetics. Furthermore, the data can be a valuable resource for comparative genomics and future research in this novel species.


Introduction
The diverse Gram-negative genus Pseudomonas contains species isolated from varied habitats, plants, animals, and people. It is a gram negative, aerobic, rod-shaped belonging to the gammaproteobacteria. 1 Here we describe a novel species Pseudomonas qingdaonensis isolated from a hospitalized patient in Kenya. Its genome sequencing will aid in our understanding of this pathogen's biology. The draft genome assembled here will enable more in-depth studies to be conducted in specific genome sections or genes that promote the novel Pseudomonas species reported here in the colonization of hospitalized patients.

Sample collection
The Pseudomonas qingdaonensis was isolated from a hospitalized patient at Kenyatta National hospital in Kenya. A stool sample was collected from the hospitalized patient and in the laboratory, it was cultured on MacConkey agar (HiMedia Lab, Mumbai, India) at 37°C for 18 hours. The colonial morphology on MacConkey agar plate, catalase, oxidase and gram staining procedure tests were used in identification. 2 The colonies on MacConkey were flat, 2-3mm, smooth colonies with an irregular leafy margin. Under the microscope after gram staining procedure, gram negative rods measuring 0.6-0.8 μm were identified. On the biochemical tests, the colonies isolated were catalase positive, and oxidase positive. Confirmation of the ID of Pseudomonas species was done using the VITEK ® 2 (Biomerieux, France). Before loading isolates into the VITEK ® 2, bacterial suspensions were done by emulsifying the isolates in 0.5% saline and standardizing turbidity to 0.5 McFarland's using a densitometer. The suspension was used for species ID and AST in the VITEK ® 2 using Gram-negative ID cards (ID-GN 21341) and results analyzed according to Clinical and Laboratory Standards Institute guidelines. 3 DNA extraction and library preparation A single colony was sub-cultured on nutrient agar (HiMedia Lab, Mumbai, India) at 37°C for 18 hours. Colonies from nutrient agar were resuspended in 400 ul of phosphate buffered saline and then extracted using ISOLATE II Genomic DNA Kit (Meridian Bioscience Inc, London, UK). The DNA was eluted with pre-warmed nuclease free water in a total volume of 50 ul. Sequencing library preparation was done using the Oxford Nanopore genomic sequencing kit SQK-LSK109 (ONT, UK) and Native Barcoding expansion kits (EXP-NBD104, EXP-NBD114) as per the manufacturer's instructions. Sequencing was performed using the MinKNOW software (ONT, United Kingdom, Oxford) with live Base calling turned on. The quality score of the FAST5 and subsequent FASTQ files produced by MinKNOW was set at 7.

Ethics declarations
The Kenyatta National Hospital -University of Nairobi (KNH-UON) Ethics and Research Committee was sought for approval and approved under Ref no. P391/07/2020. Signed informed consent, approved under Ref no. P391/07/2020, was obtained from the participant and the project followed ethical principles and guidelines for research involving human subjects.

Results
The total length of the Pseudomonas qingdaonensis was 8,857,650 base pairs long consisting of 5(five) contigs. The N50 contig was 3332773 and the GC% content was 64.03% (Table 1). The total coverage of the draft genome was 51X.
The draft genome was submitted to NCBI GenBank repository under the accession number JANWGM000000000.1. The genome annotation done using PGAP showed the presence 6,274 genes with 3,090 being coding genes, 19 rRNAs and 70 tRNAs ( Table 2).  The draft sequence of Pseudomonas qingdaonensis was submitted to the PubMLST website for multilocus sequence typing (https://pubmlst.org/) to generate an in-silico MLST profile. Table 3 show the allelic matches from PUBMLST.

Limitations
Comparative analyses were not undertaken, and more research is needed to identify Pseudomonas qingdaonensis relationship to other Pseudomonas qingdaonensis isolates globally.