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BMC Microbiology

, 19:126 | Cite as

Genomic analysis of methicillin-resistant Staphylococcus aureus strain SO-1977 from Sudan

  • Mohamed S. Ali
  • Nurulfiza M. Isa
  • Faisal M. Abedelrhman
  • Tahani B. Alyas
  • Sara E. Mohammed
  • Abdallah E. Ahmed
  • Zainab S. A. Ahmed
  • Nyok-Sean Lau
  • Mohamed I. Garbi
  • Abdullah Al-Ashraf Amirul
  • Almeen O. Seed
  • Rihab A. Omer
  • Sofia B. MohamedEmail author
Open Access
Research article
Part of the following topical collections:
  1. Microbial genetics, genomics and proteomics

Abstract

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is known as a leading cause of morbidity and mortality. Investigation of the MRSA’s virulence and resistance mechanisms is a continuing concern toward controlling such burdens through using high throughput whole Genome Sequencing (WGS) and molecular diagnostic assays. The objective of the present study is to perform whole-genome sequencing of MRSA isolated from Sudan using Illumina Next Generation Sequencing (NGS) platform.

Results

The genome of MRSA strain SO-1977 consists of 2,827,644 bp with 32.8% G + C, 59 RNAs and 2629 predicted coding sequences (CDSs). The genome has 26 systems, one of which is the major class in the disease virulence and defence. A total of 83 genes were annotated to virulence disease and defence category some of these genes coding as functional proteins. Based on genome analysis, it is speculated that the SO-1977 strain has resistant genes to Teicoplanin, Fluoroquinolones, Quinolone, Cephamycins, Tetracycline, Acriflavin and Carbapenems. The results revealed that the SO-1977, strain isolated from Sudan has a wide range of antibiotic resistance compared to related strains.

Conclusion

The study reports for the first time the whole genome sequence of Sudan MRSA isolates. The release of the genome sequence of the strain SO-1977 will avail MRSA in public databases for further investigations on the evolution of resistant mechanism and dissemination of the -resistant genes of MRSA.

Keywords

Methicillin -resistance Staphylococcus aureus (MRSA) Whole genome sequencing Antibiotic resistant genes Genome annotation Sudan 

Abbreviations

CDS

Coding sequences

MRSA

Multidrug-resistant Staphylococcus aureus

NGS

Next-generation sequencing

WGS

Whole genome sequencing

RAST

Rapid Annotation using Subsystem Technology

Background

Staphylococcus aureus (S. aureus) is a human pathogen known to cause both nosocomial and community-acquired infections [1]. It has been identified, among other classes of bacteria, resistant against some antibiotics. One of the emerged resistant strain of S. aureus is Methicillin-resistant Staphylococcus aureus (MRSA) that is the leading cause of life-threatening infections even in countries with advanced health surveillance and maintenance systems [1, 2]. In Sudan, MRSA’s incidence rate has increased dramatically and has been reported to be associated with wound infection constituting substantial sources of the high morbidity and mortality rate [3]. Such emergence of resistant strains is due to the overuse of not developed antibiotics that ultimately makes real challenges at treatment. Therefore, there is an urgent need to uncover the genetic basis of their virulence and resistance mechanism for better understanding as well as addressing potential effective drug targets. Over the last decades, Whole-genome sequencing (WGS) technologies witnessed large volumes of produced data including mutant genes, cancer-causing genes and genes predisposing for certain diseases. Moreover, the advanced bench-top sequencers technique, applied in regular clinical laboratories [4] may result in enormous diagnostic developments and challenges [5]. Genomic materials of S. aureus strains have been studied to understand the mechanisms and virulence factors responsible for staphylococcal antibiotic resistance. The premier S. aureus genomes sequenced were; MRSA strains N315 and Mu50 [6] followed by other nine strains [7, 8]. The studies revealed that the length of staphylococcal genomes is about 2.8 Mbp with low GC content. The regions of staphylococcal genomes are well conserved, with many massive sequence blocks showing high variability [8]. Although a considerable number of the MRSA resistant to antimicrobials including Methicillin, Ofloxacin, Penicillin, Amikacin, and Vancomycin are reported in Sudan [9], the molecular investigations that help in understanding the mechanism of MRSA epidemics at the whole genome level are yet limited. The present study aims to analyse the whole genome sequence (WGS) of SO-1977 strain and subsequently evaluates the genomic diversity and genotypic prediction of the antimicrobial resistance of MRSA isolated from a patient in Sudan.

Results

Genome project history

The genome sequences of SO-1977 strain were deposited in GenBank® (WGS database). The result was summarized in (Table 1).
Table 1

Project Information

Property

SO-1977

Finishing quality

Complete

Libraries used

2 × 250 bp

Sequencing platforms

Illumina MiSeq

Fold coverage

122.26x

Assembly Method

SPAdes v. 3.9.0

GenBank ID

NFZY00000000

GenBank date of Release

27-JUL-2017

BIOPROJECT

PRJNA385553

BioSample

SAMN06894057

Locus Tag

CA803

Source Material Identifier

Wound

Project relevance

Medical

Genomic features of strain SO-1977

As can seem from the data in Table 2, the draft genome sequence of S. aureus strain SO-1977 consisted of 2,827,644 bp with a 32.8% GC. The number of predicted coding sequences (CDS), tRNAs and rRNAs was 2629, 51 and 4 respectively. The final assembly contained 151 contigs with N50 of 62,783 bp length. The largest contig assembled was 146,886 bp length.
Table 2

Nucleotide and gene content’s levels of the MRSA SO-1977 genome

Attribute

Value

Genome size (bp)

2,827,644 bp

DNA G + C content

32.8%

Number of Contigs

151

N50

62,783 bp

rRNA genes

4

tRNA genes

51

ncRNAs

4

Protein-coding genes

2629

Pseudo Genes (total)

129

Pseudo Genes (frameshifted)

62

Pseudo Genes (incomplete)

32

Pseudo Genes (internal stop)

50

Pseudo Genes (multiple problems)

13

Genes assigned to SEED

1698

Genome annotation using RAST (Fig. 1)

Whole-genome annotation of MRSA strain SO-1977 on RAST server revealed a total of 1970 genes belonging to 26 subsystems such as Cofactors, Vitamins, Prosthetic Groups, Pigments, Cell Wall and Capsule and Virulence, Disease and Defense. The graphical circular map of the SO-1977 genomes was shown in Fig. 2.
Fig. 1

Summary of annotation for MRSA strain SO-1977 based on RAST subsystem

Genes involved in virulence, disease and defence

Result revealed that 83 genes encoded for virulence, disease, and defence, 28 genes were annotated to be responsible for adhesion, 32 for antibiotic resistances and toxic compounds, 14 for Bacteriocins, ribosomally synthesized antibacterial peptides and 9 for invasion and intracellular resistance (Fig. 3). Some of these genes which coding functional proteins are Fibronectin binding protein, Chaperonin, Two-component response regulator BceR, Folylpolyglutamate synthase, Acetyl-coenzyme A, Carboxyl transferase beta chain, Colicin V production protein, MerR family, Multidrug resistance protein, Mercuric ion reductase and Arsenate reductase. The category of the cell wall and capsule system of peptidoglycan biosynthesis revealed that two genes have a relationship with conferring Methicillin resistance while one gene was related to Penicillin resistance.
Fig. 2

Circular map of the chromosome of the S. aureus SO − 1977. The innermost ring represents the SO − 1977 chromosome. The second ring (in black) plots the G + C content of the reference, followed by its G + C skew (in purple/green)

Fig. 3

Bar diagram. Genes involved in category virulence disease and defense

Phages, prophages, transposable elements, plasmids (Table 3)

The analysis revealed that 35 genes are encoding for Phages, Prophages, Transposable elements, Plasmid of which 33 were annotated to be responsible for Phages, Prophages and Pathogenicity islands.
Table 3

Systems included Phages, prophages, transposable elements, plasmids category

Category

Subcategory

Subsystem

Role

Phages, Prophages, Transposable elements, Plasmids

Phages, Prophages

Phage tail proteins

Phage tail protein/ Phage tail length tape-measure protein

Phage replication

Single stranded DNA-binding protein/ Phage replication initiation protein/ DNA polymerase III alpha subunit/ DNA helicase, phage-associated

Phage packaging machinery

Phage DNA packaging/ Phage terminase, small subunit/ Phage terminase, large subunit/ Phage portal protein

Phage capsid proteins

Phage capsid and scaffold/ Phage head maturation protease/ Phage major capsid protein/ Phage capsid protein

Phage lysis modules

Phage lysin, N-acetylmuramoyl-L-alanine amidase/ Phage holin

Pathogenicity islands

Listeria Pathogenicity

Island LIPI-1 extended

Phosphatidylinositol-specific phospholipase

Zinc metalloproteinase precursor

Resistant genes based comparative genomic analysis (Table 4)

The Genome annotation and comparison results by RSAT server have shown that SO-1977 strain possesses 29 genes that may be related to multi-drug resistance and the comparison between MRSA strains was shown that 23 resistant genes were present in all strains, two genes were only found in SO-1977 strain conferring resistance against Tetracycline. Furthermore, The SO-1977 strain was the only one having the norA gene providing resistance against Quinolone beside other six genes of the family MarR. Four genes that are responsible for anti- Methicillin resistance (LytH, MecI, Mec and MurE) were only found in MRSA252 strain. Also the results have shown that MRSA252 and MSSA476 are sharing a single common gene for anti-Methicillin resistance (HmrB).
Table 5

List of 16S rRNA

Strain

Accession no.

Seq match

S. aureus

L37597.1

1

S. aureus MPU99

AB353073.1

1

S. aureus SA1

AB305019.1

1

S. aureus ATCC 14458

DQ997837.1

1

S. aureus subsp. anaerobius DSM 20714

AY688031.1

1

S. aureus K16–13

AY859409.1

1

S. aureus1

AY144447.1

1

aSeq match score, the number of (unique) 7-base oligomers shared between your sequence and a given RDP sequence divided by the lowest number of unique oligos in either of the two sequences

Phylogenetic analysis of nucleotide sequence of strain SO-1977

Result on the phylogenetic of 16S rRNA (MK713975) showed that the SO-1977 strain has the highest similarity with different S. aureus strain (Table 5) (Fig. 4).
Table 4

Summary of CDSs annotated to antibiotic resistance between SO-1977, MRSA252 and MSSA476 strains

No

SO-1977

MRSA252

MSSA476

Seed subsystem

Seed function

Length (bp)

Contig number

1

 

Peptidoglycan Biosynthesis

Methicillin resistance determinant MecA, transpeptidase

2007

Contig 000034

2

Multidrug Resistance, 2-protein version Found in Gram-positive bacteria

Multidrug resistance protein [function not yet clear]

648

Contig 000037

3

Multidrug Resistance, 2-protein version Found in Gram-positive bacteria

Membrane component of multidrug resistance system

1932

Contig 000037

4

Multidrug Resistance, 2-protein version Found in Gram-positive bacteria

TetR family regulatory protein of MDR cluster

555

Contig 000037

5

  

Tetracycline resistance, ribosome protection type

Tetracycline resistance protein TetM

1920

Contig 000042

6

  

Tetracycline resistance, ribosome protection type

Translation elongation factor G

2082

Contig 000014

7

Teicoplanin-resistance in Staphylococcus

Teicoplanin resistance associated membrane protein TcaA

1209

Contig 000002

8

Teicoplanin-resistance in Staphylococcus

Teicoplanin resistance associated membrane protein TcaB

1383

Contig 000002

9

Teicoplanin-resistance in Staphylococcus

Teicoplanin-resistance associated HTH-type transcriptional regulator TcaR

456

Contig 000002

10

  

Quinolone resistance protein norA

none

1167

Contig 000007

11

 

Bicyclomycin resistance protein TcaB

none

1212

Contig 000002

12

Transcriptional regulator, MarR family

None

456

Contig 000003

13

Transcriptional regulator, MarR family

None

420

Contig 000005

14

Transcriptional regulator, MarR family

None

210

Contig 000043

15

Transcriptional regulator, MarR family

None

435

Contig 000030

16

Transcriptional regulator, MarR family

None

441

Contig 000001

17

Transcriptional regulator,MarR family

None

468

Contig 000030

18

Resistance to fluoroquinolones

DNA gyrase subunit B

2403

Contig 000010

19

Resistance to fluoroquinolones

DNA gyrase subunit A

2661

Contig 000011

20

Resistance to fluoroquinolones

Topoisomerase IV subunit B

1998

Contig 000010

21

Resistance to fluoroquinolones

Topoisomerase IV subunit A

2403

Contig 000010

22

Beta-lactamase

Beta-lactamase

1500

Contig 000002

23

Beta-lactamase

Beta-lactamase repressor BlaI

381

Contig 000067

24

Multidrug Resistance Efflux Pumps

Acriflavin resistance protein

3168

Contig 000001

25

Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps

Multidrug Resistance Efflux Pumps

1356

Contig 000005

26

 

Methicillin resistance in staphylococci

HmrB protein involved in methicillin resistance

1600

NC_002952

27

Methicillin resistance in staphylococci

Beta-lactamase repressor BlaI

381

Contig 000067

28

Methicillin resistance in staphylococci

FmhA protein of FemAB family

1251

Contig 000002

29

 

 

Methicillin resistance in staphylococci

LytH protein involved in methicillin resistance

1600

NC_002952

30

 

Methicillin resistance in staphylococci

Methicillin resistance regulatory sensor-transducer MecR1

1600

Contig 000034/ NC_002952

31

 

 

Methicillin resistance in staphylococci

Methicillin resistance repressor MecI

1600

NC_002952

32

 

 

Methicillin resistance in staphylococci

Transposase for insertion sequence-like element IS431mec

1296

NC_002952

33

 

 

Methicillin resistance in staphylococci

UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase

1600

NC_002952

34

Methicillin resistance in staphylococci

Beta-lactamase regulatory sensor-transducer BlaR1

1089

Contig 000067

Fig. 4

Neighbour-joining tree based on 16S rRNA gene sequences showing the Phylogenetic relationship between Staphylococcus aureus strain SO-1977 relative to other type strains within the Staphylococcus aureus in database. Bootstrap values (expressed as percentages of 100 replications) less than 50% are hidden

Discussion

The present study reported the first genome sequence of S. aureus (MRSA) isolated from Sudan to have phylogenetic allocation using the 16S rRNA gene to represent the evolutionary relationships of the bacteria. In this study, the phylogenetic analysis of the complete 16S rRNA gene sequence of strain SO-1977 (MK713975) has shown that the strain should be assigned to the genus Staphylococcus. The annotated draft genome sequence of SO-1977 strain was 2827,644 bp length containing 2629 coding sequences (CDS). Moreover, the WGS data was used to investigate antimicrobial resistance and virulence mechanism. The multi-drug resistance of this isolate might be generated by the ability of these bacteria to accumulate multiple genes on the resistance (R) plasmids coding for a single drug resistance within a single cell or by the increased expression of genes that code for multi-drug efflux pumps, extruding a wide range of drugs [10]. In this study, S. aureus (MRSA) isolated from Sudan has been demonstrated to possess different resistance mechanisms which can be attributed to the use of resistant genes TcaR, TcaA, TcaB, TetR, TetM, PBP2a (MecA), or by secretion of enzymes (DNA gyrase subunit A, DNA gyrase subunit B, Topoisomerase IV subunit A, Topoisomerase IV subunit B and Beta-lactamase repressor) allowing it to use the efflux pump mechanism. In addition, six putative MarR family transcriptional regulators in the SO-1977 genome were identified. These were recognised as a widely conserved group of multiple antibiotic resistance regulators that respond to a wide range of antibiotics [11]. The MRSA characteristic phenotype is due to the presence of mecA, which encodes a penicillin-binding protein (PBP), PBP2a, with reduced affinity for b-lactams. MecA is embedded in a large heterologous chromosomal cassette, the SCCmec element. Some MRSA strains carry upstream to the mecA gene such as the regulatory genes mecI-mecR1 that encoding for a repressor and a sensor/inducer of the mecA expression, respectively [12]. In this study, MecA and MecR1 were found in SO-1977 and MRSA252, while the mecI was found only in MRSA252. This result suggested that the existence of yet unidentified additional determinants involved in the transcriptional control of mecA gene and point to a revision of the mecA regulatory mechanism in MRSA SO-1977 strain. The antibiotic sensitivity tests demonstrated that the isolate is resistant to discsoxacillin and cefoxitin. The result of the WGS confirmed the resistance of the isolate to the antibiotics and expanded it to include Teicoplanin, Fluoroquinolones, Quinolone, Cephamycins, Tetracycline, Acriflavin and Carbapenems. Such a result should be considered while planning an effective treatment protocol. The antibiotic-resistant genes of SO-1977, MRSA252 and MSSA476 revealed that the SO-1977 strain isolated from Sudan is complicated and has a wide range of cross-antibiotic resistance.

Conclusion

The current study is the first of its kind in Sudan to give an insight of an important antibiotic resistant bacterial strain, MRSA SO-1977. The SO-1977 strain is resistant to Teicoplanin, Fluoroquinolones, Quinolone, Cephamycins, Tetracycline, Acriflavin and Carbapenems. This study strongly suggests that other yet unidentified determinants are involved directly or indirectly in the transcriptional control of the mecA gene in SO-1977 strain. The SO-1977 strain has a wide range of antibiotic resistance compared to other strains. The whole genome of SO-1977 strain can provide a genetic background of virulence, antibiotic resistance and Phages of the MRSA species in Sudan.

Methods

Sample preparation

A wound swab specimen was collected from a patient at Soba Hospital, Khartoum, and was inoculated in sheep blood agar and mannitol salt agar at 37 °C for 24 h. For the purpose of colonies identification, standard procedures and tests were performed including gram stain, catalase, coagulase, and DNase tests were used to identify the colonies [13]. The positive cultures for S. aureus were then suspended with a concentration similar to turbidity standard equivalent to 0.5% McFarland and streaked on Mueller-Hinton agar (MHA). Oxacillin (6μg\ml) and cefoxitin (30μg\ml) antimicrobial disc were positioned at suitable distances on the bacterial lawns on MHA at 33 °C for 24 h. The antibiotic resistance profiling of the strain against a broader range of antibiotics was not performed as a limitation of the study. The growth inhibition zones were then measured according to the standard Kirby –Bauer disc diffusion method and NCCLs guidelines using a calliper [14]. In which the revealed measurements were indicatives of resistant colonies of MRSA strain.

Genomic DNA extraction and sequencing library preparation

Bacterial DNA was extracted using Qiagen Kit following the manufacturer instructions. The concentration and purity of the resultant DNA were photo-metrically determined using a Nano-drop (Thermofisher®). About 5 μg of genomic DNA (A260/280 = 1.93) was used for library preparation and 4 nm of genomic DNA was used as an input for the Nextera XT kit (Illumina). Then samples were targeted for bar-coding using forward (N702) and reverse (N702) primers in 12 cycles of amplification in the PCR machine. Libraries were then quantified on the Bioanalyzer (Agilent Technologies) and combined with an equimolar mixture. Finally, 0.19 ng/ ml was used as an input for Next-generation sequencing (NGS) and libraries were sequenced on a single run on the Illumina MiSeq instrument (250 bp paired-end reads).

Bacterial genome sequencing and assembly

Poor-quality and adaptor-containing reads were filtered and trimmed using BBTools version 36 [15]. Good quality sequencing reads were assembled using SPAdes version 3.5.0. For the prediction of tRNA and rRNA genes, ARAGORN 1.2.34 and RNAmmer1.2 were used, respectively [16, 17]. The protein-coding genes were then predicted using Prodigal 2.60 [18] as well as their function by using BLASTN 2.2.25+ [19] and followed by detecting sequence homologs through searching for various sequence domain databases using HMMER 3.0 (http://hmmer.org/).

Genome annotation

The final draft genome sequence of S. aureus SO-1977 was used for annotation using RAST [20] and NCBI Prokaryotic Genome Annotation Pipeline [21]. The annotated genes were exported from the RAST server into an excel table and manually compared for genomic features. The antibiotic resistance genes of the S. aureus SO-1977, S. aureus MRSA252 (PRJNA265) and S. aureus MSSA476 (PRJNA116329) were retrieved from RAST server then the comparison was done [22]. The graphical circular map of the genomes was made by CGView server [23].

Phylogenetic analysis of strain SO-1977 using 16S rRNA

The 16S rRNA sequences were edited and assembled to a final length of 1545 bp, GenBank accession number (MK713975). All reference sequences of the 16S rRNA genes of S.aureus used in this study were obtained from GenBank® (https://www.ncbi.nlm.nih.gov/genbank/) using the RDP 11.5, Seqmatch: version 3 (https://rdp.cme.msu.edu/). DNA sequence alignment was performed using MUSCLE v. 3.8.31 (http://www.ebi.ac.uk/Tools/msa/muscle/) on the European Bioinformatics Institute (EBI) homepage. Once the alignment was completed, the phylogenetic tree was drowning as the evolutionary distances were computed using the Maximum Likelihood method implemented in MEGA6 version 6 [24] in all positions containing gaps and missing data were eliminated.

Sequence data access

The genomic data of this study were deposited publicly in DDBJ/ENA/GenBank® under Accession: NFZY00000000, BioProject: PRJNA385553 and Biosample: SAMN 06894057.

Notes

Acknowledgements

We thank Prof. Mohamed M.A Alnour and Ms. Somyia Kambal for reviewed the manuscript. This work was supported by National University Research Institute-National University, Sudan.

Funding

This work was supported by National university research institute / National University. The funding body had a role.

Availability of data and materials

The datasets generated during the current study are available from the corresponding author publicly available due (Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession NFZY00000000 and PRJNA385553. The version described in this paper is version NFZY01000000.

Authors’ contributions

SBM, MSA and AOS planned and directed the project. SEM and MIG collected the sample, TBA, AEA, ZSAA and FMA laboratory work and bacterial identification. SBM, RAO and NMI drafted the manuscript. SBM, N-SL and AAA performed the gene annotation and comparative genomic analysis and interpreted the results. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This research was approved by the Ethical Committee of the International University of Africa, Khartoum Sudan and written consent was obtained.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Mohamed S. Ali
    • 1
  • Nurulfiza M. Isa
    • 2
  • Faisal M. Abedelrhman
    • 1
  • Tahani B. Alyas
    • 3
  • Sara E. Mohammed
    • 4
  • Abdallah E. Ahmed
    • 1
  • Zainab S. A. Ahmed
    • 1
  • Nyok-Sean Lau
    • 5
  • Mohamed I. Garbi
    • 6
  • Abdullah Al-Ashraf Amirul
    • 5
  • Almeen O. Seed
    • 1
  • Rihab A. Omer
    • 1
  • Sofia B. Mohamed
    • 1
    Email author
  1. 1.Department of Bioinformatics and BiostatisticsNational University Research Institute- National UniversityKhartoumSudan
  2. 2.Department of Cell and Molecular Biology, Faculty of Biotechnology and Bimolecular SciencesUniversiti Putra MalaysiaSelangorMalaysia
  3. 3.Tropical Medicine Research InstituteKhartoumSudan
  4. 4.Department of microbiology, faculty of medical laboratory sciencesNational UniversityKhartoumSudan
  5. 5.Centre for Chemical BiologyUniversiti Sains MalaysiaBayan LepasMalaysia
  6. 6.Department of Microbiology, Faculty of Medical Laboratory ScienceInternational University of AfricaKhartoumSudan

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