Question

how to find lineage of Streptococcal pyogenes via Using BLAST. lineage means it's ancestor/roots.

Answer 1

Introduction

Streptococcus pyogenes, also referred to as Group A Streptococcus (GAS) for harboring Lancefield group A antigen, is a clinically important human pathogen. Despite its limited prevalence in modern times as compared to other pathogens, the myriad of infections it causes are still commonly lethal. Streptococcal infections range from localized throat infections such as tonsillitis or pharyngitis, to invasive infections such as sepsis, necrotizing fasciitis and streptococcal toxic shock syndrome (STSS). Most of the infections are seen in children aged four to seven years, with penicillin still being effectively used for treatment. Drug resistant clones however, have been increasingly reported globally , and were attributed to environmental and intracellular resistance mechanisms . Treatment complications usually arise with the use of other antimicrobial agents when the patient is allergic to penicillin.

A number of genome-encoded virulence factors such as pili, M proteins, leukocidins, streptolysins, complement inhibiting proteins, immunoglobulin-degrading enzymes, and superantigens have been detected in S. pyogenes, in addition to efflux pumps and leukocyte evasion strategies. Interestingly horizontal gene transfer (HGT) and prophage integration are common amongst S. pyogenes genomes giving them plasticity and genomic variation. These factors collectively confer additional virulence and resistance capabilities and alter the regulation of existing genes. The emm gene, encoding the M protein contains conserved, semi-conserved, and hypervariable regions, and is thus used as an epidemiological marker for GAS. emm occurrence amongst GAS strains is linked to geographic localization.

Materials and Methods

Ethical Approval

Ethical approval was not required as clinical isolates were collected and stored as part of routine clinical care. Clinical isolates and patient records/information were anonymous and de-identified prior to analysis.

Bacterial isolates and genomic DNA extraction

This study was conducted on nine S. pyogenes bacterial isolates previously collected from the American University of Beirut Medical Center (AUB-MC). The samples were recovered from throat and pus swabs of patients with streptococcal infections during the period from August 2010 to November 2011, and were chosen to cover the common emm types in the country (Table 1). The isolates were cultured overnight on Trypticase Soy Agar (TSA) (Bio-Rad, USA) medium. DNA was extracted using the Nucleospin Tissue kit (Macherey-Nagel, Germany) following the manufacturer’s instructions.

Table 1. Epidemiological Information on clinical S. pyogenes isolates. Sex Age Origin Specimen Disease Sample Name SP1 ST. Type ST-36 emm typing in (12) I 12 Virulence Factors in (13) Cys. Prot. B-Spel emm Pattern Tissue preference/ Associated Disease ThroatURT F A-C 48 Lebanon Swab. Pharyngitis throat 47 Lebanon Swab, pus Dermatitis SP2 F ST- 108 Cys. Prot. B-Spel Skin SP3 M 7 Lebanon Pharyngitis ST- Cys. Prot. B-Spel No preference/URT SP4 8 Lebanon Swab. throat Swab. throat Swab, pus Pharyngitis ST-52 Cys. Prot. B-Spel- No preference/Invasive SP5 M 35 Lebanon Dermatitis ST-28 A-C Throatinvasive SpeA-Cys. Prot. B Spel Speg SP6 F 23 Lebanon Swab, throat Pharyngitis ST. No preference/URT SP7 Pharyngitis ST-46 No preference/Invasive M F 4 Lebanon 1 Lebanon SpoA-Cys. Prot. B- ssa-smez SpeA-Spessa SP8 Swab. throat Swab. throat Swab. throat Pharyngitis ST. 85 Skin Skin SP10M 7 Lebanon Pharyngitis ST- 118 Cys. Prot. B-Spell- Spek-smez E No preference doi:10.1371/journal.pone.0168177.1001

Genome sequencing

DNA extracted from each S. pyogenes isolate (50-ng/sample) was prepared for sequencing with the use of the Nextera XT DNA Sample Prep Kit (Illumina). Clean up was performed using the AMPure XP PCR purification beads (Agencourt, Brea, CA, USA). The resulting individual DNA libraries with fragment sizes ranging from 500–1000 bp were quantified by quantitative PCR on a CFX96 (Bio-Rad, USA) in triplicate at two concentrations, 1:1000 and 1:2000, using the Kapa library quantification kit (Kapa Biosystems, Woburn, MA, USA). Based on the individual library concentrations, equimolar pools of the indexed libraries were prepared at a concentration of at least 1 nM using 10 mM Tris-HCl (pH 8.0) and 0.05% Tween 20. Pooled paired-end libraries were sequenced to a read length of at least 250 bp.

Genome assembly

De novo assembly of the sequenced genomes was done using A5 assembler (v. 20130627) with default assembly parameters. This pipeline automates the processes of data cleaning, error correction, contig assembly, scaffolding, and quality control .

Genome annotation and gene detection

The assembled genomes were annotated using the RAST server (http://rast.nmpdr.org) that uses subsystems technology to assign gene function. RAST was also used for the identification of protein encoding genes, rRNAs, and tRNAs . The SEED viewer service from RAST in combination with the web tools provided by the Center for Genomic Epidemiology (CGE) website (www.genomicepidemiology.org) were used to generate a detailed list of the genetic elements in question. The ResFinder 2.1 web server was used to identify acquired antimicrobial resistance genes present on the bacterial genome . Due to ResFinder’s inability to confirm the functional integrity, and levels of gene expression and resistance arising to acquired mutations in housekeeping genes, a hybrid resistance profile was generated using the ResFinder hits in addition to phenotypical data from previously published studies. The VirulenceFinder 1.2 service from the same website was used to identify additional virulence-specific genes . The PathogenFinder 1.1 service from CGE, was used to obtain an overview of the genomic pathogenic gene families .

Multi-locus sequence typing (MLST)

MLST typing of the isolates was carried out using CGE’s MLST 1.7 server to detect sequence polymorphisms within the gki, gtr, muri, muts, recp, xpt, and yqil genes .

Phage and mobile element detection

Phage detection was done using the publicly available Phage Search Tool (PHAST) (http://phast.wishartlab.com/index.html) . This tool provides the closest identity match for detected phages in addition to their site of integration. Putative insertion elements were double checked using BLASTx with an identity threshold of 80%. Putative phage insertion sequences were then annotated using RAST in order to determine the genes they encode.

Phylogenetic analysis

To determine the phylogenetic relatedness a concatenated marker gene maximum-likelihood tree was constructed using a number of S. pyogenes reference genomes chosen based on BLAST similarity results, clonal complexes and sequence types (MGAS6180 CP000056, MGAS10394 CP000003, M1476 AP012491, M1GAS SF370 NC002737, NZ131 CP000829, A20 CP003901, 7F7 PRJNA238516, and S. pneumoniae R6 AE007317 as an outlier strain). The genomes were first processed with PhyloSift , the tree was then constructed using FastTree , visualized and edited with Dendroscope . Pairwise alignment and visualization of our selected genomes with the respective reference strains was achieved through the Mauve aligner using defaults settings.

Results and Discussion

Sequencing resulted in an average of 2,503,465 paired-end reads per isolate, with the average being 1,976,732 high-quality reads following quality trimming and error correction. The average sequence coverage of the whole genomes was 293X and the minimum sequence coverage was 85X for the SP6 isolate. The average N50 for the assemblies was 196,439 bp with the lowest being 98,990 bp for SP7. The initial assemblies resulted in an average of 77 contigs per isolate all of which greater or equal to 500 bp in length. During scaffolding, some contigs were merged based on short overlaps and read-pair information, yielding a reduced final average of 70 contigs per isolate. The complete details and statistics of the sequencing and assembly results for each isolate are shown in Tables 2 & 3 .

SP3 1,745,842 SP4 1,906,369 SP5 1,813,544 21 28 26 Genome size (bp) Number of contigs (>= 500 bp) Average contig size (bp) Longest contig size (bp) GC content (mol %) N50 (bp) doi:10.1371/journal.pone.0168177.t002 SP1 1,925,871 155 12,425 343,437 38.4 209,002 SP2 1,727,943 29 59,584 653,370 38.4 280,940 83,135 659,470 38.4 167,926 68,085 249,942 38.2 205,587 69,752 752,626 38.4 308,655 SP6 1,733,546 19 91,239 674,975 38.4 116,374 SPZ 1,953,601 158 12,365 207,328 38.4 98,990 SP8 1,917,411 186 10,309 250,346 38.5 104,600 SP10 1,771,196 12 147,600 781,011 38.4 275,797

SP3 1725 SP1 1991 22 1907 SP2 1694 26 1669 65 SP4 1905 24 1923 # of predicted genes # of pseudogenes # of predicted potein-coding genes # of tRNA genes # of rRNA genes # of subsystems doi:10.1371/journal.pone.0168177.6003 SP5 1832 18 1824 57 9 319 SP7 2041 31 1937 27 1711 56 SP6 1698 30 1692 54 4 314 SP8 1971 37 1866 56 10 322 SP10 1740 34 1740 56 4 317 56 54 56 7 6 311 6 317 5 316 317 313