Whole genome sequence data of the multidrug-resistant Proteus mirabilis-MN029, a model bacterial strain for the evaluation of antibacterial activity from selected native plant extracts

Abstract

Antimicrobial resistance remains a significant global and One Health threat, owing to the diminishing effectiveness of antibiotics against rapidly evolving multidrug-resistant bacteria, and the limited innovative research towards the development of new antibiotic therapeutics. In this article, we present the whole-genome sequence data of Proteus mirabilis-MN029 obtained from highly accurate long-read PacBio^Ⓡ HiFi technology. The antibacterial activities of the selected African native plant species were also evaluated using the disk diffusion method. Acquired antibiotic resistance genes and chromosomal mutations corresponding to antibiotics of clinical importance were identified from genomic data. Using ethlyl acetate as solvent, Pterocarpus angolensis leaf extracts showed the most promising antibacterial effects against Proteus mirabilis-MN029. These datasets will be useful for future experimental research aimed at designing new antibacterial drugs from plant extracts that are effective alone or in combination with existing antibiotics to overcome multidrug-resistance mechanisms.

Specifications Table

Subject	Applied Microbiology/ Genomics
Specific subject area	Genomics of multidrug-resistant bacteria and antimicrobial activity of native plants
Data format	Assembled data in FASTA format.
Type of data	Table and Figure
Data collection	Multidrug resistant P. mirabilis-MN029 strain was isolated from polluted soil sample originating from a rural environment in Palapye, Botswana. The strain was sequenced using long read PacBio HiFi Technology. The selected medicinal plants Pterocarpus angolensis, Aloe zebrina and Aloe littoralis were collected from Tsodilo and Mobolwe geographical areas in Botswana. P angolensis: 18°48’34.5”S 21°43’19.4.E, Tsodilo; A. zebrina: 21°50’37.1”S 28°43’59.6.E, Mabolwe; A. littoralis: 21°51’19.2”S 28°44’10.6.E, Mabolwe . The minimum inhibitory concentration and antibacterial activity of crude extracts from the medicinal plants against the sequenced strain was conducted using microdilution and disk diffusion methods.
Data source location	Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology, Palapye, Botswana
Data accessibility	Repository name: National Centre for Biotechnology Information (NCBI) Data identification number: Bioproject Accession Number: PRJNA860342 Direct URL to data: http://www.ncbi.nlm.nih.gov/bioproject/860342 Nucleotide Sequence Accession: NZ_CP102086.1

1. Value of the Data

• •Whole genome sequence data will offer a comprehensive understanding of the antimicrobial resistance mechanisms, virulence factors and drug targets in P. mirabilis, which will be beneficial to the healthcare providers and the scientific community.
• •The data provide an advanced understanding of the molecular/genotypic characteristics of P. mirabilis, and the associated phenotypic features.
• •The data can be exploited in silico and used concurrently to support in vitro investigations of plant secondary metabolites against drug resistance mechanisms, leading to improved drug development.

2. Background

The escalating prevalence of multidrug-resistant (MDR) bacterial strains poses a One Health threat, negatively impacting the health of humans, animals, and their immediate environment. In an effort to address this global antimicrobial resistance (AMR) problem, we shifted our research focus toward exploring native and natural African plant-derived compounds as alternative therapeutics against MDR bacteria [1]. Next-generation sequencing (NGS) technology offers greater opportunities towards expanding our knowledge of the genome structure and functional dynamics of MDR bacteria and could lead to novel scientific discoveries. We have recently evaluated and adopted the use of NGS approaches, and successfully applied different NGS techniques (Illumina short-read and Nanopore long-read sequencing) in Botswana in continued efforts to demonstrate their practicality and sustainability in low resourced settings [[2], [3]]. Parallel to our antimicrobial drug discovery research focus, we considered the PacBio^Ⓡ HiFi long-read sequencing method to obtain a whole genome sequence (WGS) data and to accurately identify and characterize the MDR strain. The identified strain Proteus mirabilis-MN029 was selected as a model bacterium for evaluation of antibacterial activity of selected native plant species because of its origin, clinical relevance, phenotypic and genomic characteristics. Proteus mirabilis is a rod-shaped, motile and Gram-negative opportunistic pathogen, that is widely distributed in the natural environment and known for causing urinary tract infections (UTIs). P. mirabilis infections pose significant health risks, these include sepsis, kidney injuries and complications especially from frequent antibiotic use [4].

3. Data Description

This article describes the WGS dataset of potentially pathogenic and MDR Gram-negative bacteria, and its susceptibility to selected antibiotics and crude extracts from native plants. The complete genome of the MDR strain is a single chromosome, spanning 3,988,563 base pairs with an average G+C content of 38.5% and encompasses 3,649 protein coding sequences (CDS). Additionally, the genome has 84 transfer RNA (tRNA) genes and 22 ribosomal RNA (rRNA) genes. The MDR bacterial strain was identified as Proteus mirabilis based on the genomic and phylogenetic analysis of WGS data. Genome annotation of P. mirabilis-MN029 reveal several CDS homologous to known proteins conferring AMR phenotypes, virulence factors, drug targets and transporters (Fig. 1). The notable acquired antibiotic resistance genes; macA, macB (macrolide), tet (J) (tetracycline, Dfr (trimethoprim) and catA (chloramphenicol) were predicted, along with several chromosomally encoded resistance genes, associated with multidrug-resistance mechanisms (Tables 1, 2).

Fig. 1.

Genome analysis of P. mirabilis-MN029. (A) Circular graphical display of the distribution of the genome annotations; Outer to inner rings correspond to the chromosome, Coding sequences (CDS) on the forward strand (CDS-Fwd), CDS on the reverse strand (CDS-Rev), Non-coding sequences (Non-CDS), CDS with homology to known antimicrobial resistance (AMR) genes, virulence factors (VF), transporters and drug targets. (B). Identification and phylogenetic analysis of the MDR bacterial strain.

Table 1.

Antibiotic susceptibility of P. mirabilis-MN029 analysed using agar dilution and the Kirby Bauer disk diffusion methods.

Antibiotic Class	Antibiotic agar dilution	Antibiotic disk	Phenotype
Penicillin	AMX AMP PEN G	AML25 AP10 PG10	R R R
Aminoglycoside	AMK KAN GEN	– K30 –	S S R
Tetracycline	TET	TE30	R
Rifamycin	RIF	RD30	S
Trimethoprim	TMP	W5	R

Keys: R, resistant; S, susceptible; –, not tested; Amoxicillin (AMX, AML25), Ampicillin (AMP, AP10), Penicillin G (PEN G, PG10), Amikacin (AMK), Kanamycin (KAN, K30), Gentamicin (GEN), Tetracycline (TET, TE30), Rifampicin (RIF, RD30), Trimethoprim (TMP, W5)

Table 2.

Summary of the AMR genes predicted in the P. mirabilis-MN029 genome and the corresponding AMR mechanisms.

AMR Mechanism	Antimicrobial Resistance Genes
Antibiotic inactivation enzyme	CatA1/CatA4 family
Antibiotic target in susceptible species	Alr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, inhA, fabI, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, S12p
Antibiotic target protection protein	BcrC
Efflux pump conferring antibiotic resistance	AcrAB-TolC, AcrZ, EmrAB-TolC, MacA, MacB, MdtABC-TolC, MdtL, MexHI-OpmD, SugE, Tet(J), TolC/OpmH
Gene conferring resistance via absence	gidB
Protein altering cell wall charge conferring antibiotic resistance	GdpD, PgsA
Regulator modulating expression of antibiotic resistance genes	AcrAB-TolC, EmrAB-TolC, H-NS, OxyR

This article further presents the results on the antibacterial activity of crude extracts from different parts of the selected native plant species; Pterocarpus angolensis, Aloe zebrina, and Aloe littoralis against the model P. mirabilis strain MN029. All plant part extracts of A. zebrina, the leaf crude extracts of A. littoralis and the seed extracts of P. angolensis obtained using different solvents showed no inhibition of P. mirabilis-MN029, as did all plant parts extracted using hexane as a solvent. The largest inhibition zone relative to the positive control (kanamycin, 30 µg) was observed at the highest concentration of 40 mg/ml across all plant species, with a significant difference only observed for the A. littoralis ethyl acetate roots extract (Fig. 2). No significant difference was observed in the zones of inhibition at 20 mg/ml for P. angolensis methanol root extracts or at concentrations up to a minimum of 5 mg/ml for P. angolensis ethyl acetate leaf extracts. Ethlyl acetate P. angolensis leaf extracts showed promising inhibitory effects at all tested concentrations with the minimum inhibitory concentration further confirmed up to 2.5 mg/ml.

Fig. 2.

Zones of inhibition shown by different crude extracts at different concentrations against P. mirabilis-MN029. Disk diffusion was performed in triplicate, and the error bars represent the standard error of the mean (SEM). ∗ P < 0.05 compared with the control, ∗∗ P < 0.005 in comparison to control, ∗∗∗ P < 0.001 in comparison to the positive control, ∗∗∗∗ P < 0.0001 in comparison to the control and ns means there is no significant difference in comparison to the control. Keys: dH2O; distilled water, PC is positive control (kanamycin, 30 µg).

4. Experimental Design, Materials and Methods

4.1. Bacterial Isolation and Multidrug-Resistance Screening

The P. mirabilis-MN029 strain was isolated on MacConkey agar along with other strain collections originating from wastewater-contaminated soil samples. The agar dilution method was used to test for resistance to different antibiotics with the following referenced breakpoint concentrations: ampicillin (32 µg/mL), gentamicin (16 µg/mL), tetracycline (16 µg/mL), amikacin (64 µg/mL), rifampicin (30.7 µg/mL), erythromycin (8 µg/mL), penicillin (16 µg/mL) and amoxicillin (32 µg/mL), as previously described by Brooks et al. [2]. The Kirby-Bauer disk diffusion method, as per the Clinical Laboratory Standards Institute (CLSI), was used to further confirm the resistance pattern of the isolates [5]. The antibiotic disk panel comprised of Amoxicillin (25 µg), Ampicillin (10 µg), Penicillin G (10 µg), Amikacin (30 µg), Kanamycin (30 µg), Tetracycline (10 µg) Rifampicin (30 µg) and Trimethoprim (5 µg).

4.2. Genomic DNA Extraction, Whole Genome Sequencing and Bioinformatics Analysis

Genomic DNA extraction from the MDR strain was carried out using a Zymo Research Fungal/Bacterial Miniprep kit [6], following the manufacturer's instructions. The extracted DNA was then quantified and assessed for purity using a NanoDrop spectrophotometer and sent for high-throughput sequencing at Inqaba Biotec, South Africa. WGS was conducted using a SMRTbell^Ⓡ prep kit 3.0 (PacBio) following the manufacturer's instructions. In preparation for long-read sequencing, the gDNA was sheared by a two-cycle shearing method to achieve target size distribution of approximately 15 kb -18 kb using the Megaruptor 3 system. SMRTbell libraries were prepared using PacBio's Microbial Multiplexing workflow [7]. The resulting libraries were quantified using Qubit HS dsDNA Assay and qualified on a TapeStation using Genomics Screen Tape. Libraries were then prepared for sequencing following the online SMRTlink guided protocol (Binding Kit 3.2 and Control 1.0, Sequel II Sequencing plate 2.0 and SMRT Cell 8M). De novo assembly of the PacBio HiFi long-reads was conducted using the PacBio open-source SMRT Analysis software (SMRTLINK v.11.1). Bioinformatics analysis conducted using two online platforms, Bacterial and Viral Bioinformatics Resource Center (BV-BRC)(https://www.bv-brc.org/) for comprehensive genome analysis and ResFinder 4.1 (https://cge.food.dtu.dk/services/ResFinder/) for the prediction of phenotypes from genotypes [8].

4.3. Crude Extract Preparation and Minimum Inhibitory Concentration (MIC) of Plant Extracts

Plant material (fruits, seeds, leaves, bark, and roots) and crude extracts were prepared following the methods by Mohamed et al. [9], with few modifications. Briefly, the plant material was sun-dried and 30 grams per 100 ml of solvent of different polarities (n-hexane, ethyl acetate, methanol, and distilled water) was used for maceration extraction. The MIC determination was performed in a 96 well microtiter plate following a protocol outlined by Haile and Jiru [10] with extended extract concentrations (5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, 0.313 mg/mL, 0.156 mg/mL, 0.078 mg/mL and 0.039 mg/ml) and incubation time at 24 h.

4.4. Antimicrobial Activity of P. angolensis, A. zebrina and P. littoralis against P. mirabilis-MN029

The disk diffusion method was carried out according to the National Committee for Clinical Laboratory Standards as described by Mohamed et al. [9] and Haile and Jiru [10], with modifications including Kanamycin (30 µg) as a zone of inhibition positive control. The solvents: DMSO, methanol, hexane, ethyl acetate, and distilled water alone served as negative controls. Crude extracts used for the assay were 40 mg/ml, 20 mg/ml, 10 mg/ml, and 5 mg/ml.

4.5. Statistical Analysis

The mean value and standard error of the mean (mean ± SEM) were calculated from triplicate assays. Statistical significance was assessed through one-way and two-way (grouped data) analysis of variance (ANOVA) using GraphPad Prism, with significance considered for values at p < 0.05.

Limitations

Antibiotic susceptibility testing of the model bacterial strain and crude plants extractions were limited to available antibiotics and solvents.

Ethics Statement

The authors have read and followed the ethical requirements for publication in Data in Brief. The current work does not involve human subjects, animal experiments, or any data collected from social media platforms.

CRediT authorship contribution statement

Katlego P.P. Makale: Data curation, Writing – original draft, Visualization, Investigation. Motlatsi Nketsang: Data curation, Writing – original draft, Visualization, Investigation. Gaolathe Rantong: Conceptualization, Supervision, Writing – review & editing. Abdullah Makhzoum: Supervision, Writing – review & editing. Teddie O. Rahube: Conceptualization, Writing – original draft, Writing – review & editing, Supervision.

Data Availability

• Proteus mirabilis Genome sequencing (Original data) (NCBI).