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

[version 1; peer review: 2 approved]
Brian Ogoti1,2, Racheal Gachogo
https://orcid.org/0000-0002-7921-6351
3, Frank Onyambu
https://orcid.org/0000-0002-1195-8762
4,5
Brian Ogoti1,2, Racheal Gachogo
https://orcid.org/0000-0002-7921-6351
3, Frank Onyambu
https://orcid.org/0000-0002-1195-8762
4,5
PUBLISHED 21 Dec 2022
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

This article is included in the Genomics and Genetics gateway.

Abstract

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.

Keywords

Draft genome, Pseudomonas qingdaonensis

Corresponding author: Brian Ogoti
Competing interests: No competing interests were disclosed.
Grant information: This study was supported by the Centre for Molecular Biosciences and Genomics, Nairobi, Kenya [CMB_001].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright:  © 2022 Ogoti B et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Ogoti B, Gachogo R and Onyambu F. Draft genome of a clinical Pseudomonas qingdaonensis isolate from a hospitalized patient at Kenyatta National Hospital, Kenya [version 1; peer review: 2 approved]. F1000Research 2022, 11:1544 (https://doi.org/10.12688/f1000research.127935.1) First published: 21 Dec 2022, 11:1544 (https://doi.org/10.12688/f1000research.127935.1) Latest published: 21 Dec 2022, 11:1544 (https://doi.org/10.12688/f1000research.127935.1)

Abbreviations

MLST: Multilocus sequence typing

rRNA: Ribosomal RNA

tRNA: Transfer RNA

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.

Methods

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.

Bio-informatics analyses

Live base calling and adapter trimming was performed using Guppy v3.6.1 (https://nanoporetech.com). De-novo assembly of the Pseudomonas qingdaonensis was performed using UNICYCLER v0.4.9 (https://github.com/rrwick/Unicycler).4 The genome quality and assembly was contrasted and carefully selected based on the following criteria: the size of contigs, GC (%) content, N50 length, and genome coverage as determined by QUAST v5.2.0 (http://bioinf.spbau.ru/quast). Visualization of the draft assembly was done using Bandage v0.9.0 (https://rrwick.github.io/Bandage).5 Genome annotation of the draft assembly was done using the NCBI Prokaryotic Genome annotation pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/).

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.

Table 1. QUAST statistics of the Pseudomonas qingdaonensis draft genome.

AssemblyAssembly
# contigs (≥ 0 bp)5
# contigs (≥ 1000 bp)5
# contigs (≥ 5000 bp)5
# contigs (≥ 10000 bp)4
# contigs (≥ 25000 bp)4
# contigs (≥ 50000 bp)4
Total length (≥ 0 bp)5857650
Total length (≥ 1000 bp)5857650
Total length (≥ 5000 bp)5857650
Total length (≥ 10000 bp)5851293
Total length (≥ 25000 bp)5851293
Total length (≥ 50000 bp)5851293
Number of contigs5
Largest contig3332773
Total length5857650
GC (%)64.03
N503332773
N90889928
L501
L903

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).

Table 2. PGAP annotation results of the Pseudomonas qingdaonensis genome.

Genes (total)6,274
CDSs (total)6,179
Genes (coding)3,090
CDSs (with protein)3,090
Genes (RNA)95
rRNAs7, 6, 6 (5S, 16S, 23S)
complete rRNAs7, 6, 6 (5S, 16S, 23S)
tRNAs70
ncRNAs6
Pseudo Genes (total)3,089
CDSs (without protein)3,089
Pseudo Genes (ambiguous residues)0 of 3,089
Pseudo Genes (frameshifted)2,865 of 3,089
Pseudo Genes (incomplete)398 of 3,089
Pseudo Genes (internal stop)63 of 3,089
Pseudo Genes (multiple problems)228 of 3,089

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.

Table 3. PubMLST sequence query of Pseudomonas qingdaonensis.

LocusAlleleLengthContigStart positionEnd position
BACT000004 (rpsD)9850621118836701884290
BACT000005 (rpsE)16706501118801531880653
BACT000006 (rpsF)75064261671121671546
BACT000008 (rpsH)8099393118788521879244
BACT000009 (rpsI)9124393123598962360288
BACT000010 (rpsJ)7797312118720601872371
BACT000011 (rpsK)7715390118832651883654
BACT000013 (rpsM)7758357118828781883234
BACT000015 (rpsO)7632270121652382165507
BACT000016 (rpsP)720325244201542266
BACT000017 (rpsQ)6904267118767871877053
BACT000018 (rpsR)68442311670863671093
BACT000021 (rpsU)5073216117985291798744
BACT000033 (rplD)9563603118731001873702
BACT000042 (rplM)8392429123594532359881
BACT000043 (rplN)7772369118770771877445
BACT000045 (rplP)8019414118761801876593
BACT000046 (rplQ)7980387118853551885741
BACT000047 (rplR)7415351118797991880149
BACT000050 (rplU)73633121374142374453
BACT000051 (rplV)7664333118751361875468
BACT000052 (rplW)7143300118736991873998
BACT000057 (rpmB)6464237113765891376825
BACT000058 (rpmC)5619192118765931876784
BACT000059 (rpmD)5467177118806561880832
BACT000060 (rpmE)7515216116394011639616
BACT000061 (rpmF)6317183129652572965439
BACT000062 (rpmG)3770156113768371376992
BACT000063 (rpmH)5737135112512951251429
BACT000064 (rpmI)6315195132742173274411
BACT000065 (rpmJ)5114117118826301882746

Limitations

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

Data availability

Underlying data

GenBank: Pseudomonas qingdaonensis, whole genome sequence, Accession number JANWGM000000000.1: https://www.ncbi.nlm.nih.gov/Traces/wgs/JANWGM01?display=contigs

BioProject: Pseudomonas qingdaonensis strain: CMB_001, Accession number PRJNA869907: https://identifiers.org/NCBI/bioproject:PRJNA869907

BioSample: Pathogen: clinical or host-associated sample from Pseudomonas qingdaonensis, Accession number SAMN30333618: https://identifiers.org/biosample:SAMN30333618

Acknowledgements

We acknowledge the Centre of Molecular Biosciences for providing the resources to sequence the genome.

References

  • 1.  Wang MQ, Wang Z, Yu LN, et al.: Pseudomonas qingdaonensis sp. nov., an aflatoxin-degrading bacterium, isolated from peanut rhizospheric soil. Arch. Microbiol. 2019 Jul; 201(5): 673–678. PubMed Abstract | Publisher Full Text
  • 2.  Giuliano C, Patel CR, Kale-Pradhan PB: A Guide to Bacterial Culture Identification And Results Interpretation. Pharm. Ther. 2019 Apr; 44(4): 192–200.
  • 3.  Clinical and Laboratory Standards Institute: CLSI: Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Seventh Informational Supplement. CLSI Document M100-S27. [Internet]. 2017; p. 282.Reference Source
  • 4.  Completing bacterial genome assemblies with multiplex MinION sequencing. Microbiology Society. [cited 2022 Dec 7]. Publisher Full Text
  • 5.  Wick RR, Schultz MB, Zobel J, et al.: Bandage: Interactive visualization of de novo genome assemblies. Bioinformatics. 2015; 31(20): 3350–3352. PubMed Abstract | Publisher Full Text | Free Full Text

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Ogoti B, Gachogo R and Onyambu F. Draft genome of a clinical Pseudomonas qingdaonensis isolate from a hospitalized patient at Kenyatta National Hospital, Kenya [version 1; peer review: 2 approved]. F1000Research 2022, 11:1544 (https://doi.org/10.12688/f1000research.127935.1)
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Reviewer Report 02 May 2023
Aquillah Kanzi, Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, Gauteng, South Africa 
Approved
VIEWS 4
https://doi.org/10.5256/f1000research.140481.r168571
The authors sequenced and assembled a draft genome of Pseudomonas qingdaonensis isolated from a hospitalised patient. The methodology used for sequencing and assembly is technically sound. The comments and suggestions below can improve the quality of this work.

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Kanzi A. Reviewer Report For: Draft genome of a clinical Pseudomonas qingdaonensis isolate from a hospitalized patient at Kenyatta National Hospital, Kenya [version 1; peer review: 2 approved]. F1000Research 2022, 11:1544 (https://doi.org/10.5256/f1000research.140481.r168571)
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NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 03 Jan 2023
Gerald Mboowa, Department of Immunology and Molecular Biology, School of Biomedical Sciences, Makerere University College of Health Sciences, Kampala, Uganda 
Approved
VIEWS 13
https://doi.org/10.5256/f1000research.140481.r158492
Take note that this is written as Gram-negative not gram-negative throughout the entire write up.

Authors should include the following to make this work more informative:
  1. Where was sequencing done?
  2. Annotation
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Mboowa G. Reviewer Report For: Draft genome of a clinical Pseudomonas qingdaonensis isolate from a hospitalized patient at Kenyatta National Hospital, Kenya [version 1; peer review: 2 approved]. F1000Research 2022, 11:1544 (https://doi.org/10.5256/f1000research.140481.r158492)
The direct URL for this report is:
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NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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  1. Gerald Mboowa, Makerere University College of Health Sciences, Kampala, Uganda
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