Phylo-taxonogenomic reconciliations of genus Streptococcus reveals the existence of sixteen novel genomospecies

doi:10.21203/rs.3.rs-1416559/v1

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Phylo-taxonogenomic reconciliations of genus Streptococcus reveals the existence of sixteen novel genomospecies

https://doi.org/10.21203/rs.3.rs-1416559/v1

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Over two centuries, genus Streptococcus has been extensively explored in clinical and industrial settings. However, its taxonomy is based on low-resolution methods such as biochemical, housekeeping genes, or conserved sequence identity of species groups resulting in various amendments. In the current study, we have used genome similarities and phylogeny to provide a robust taxonomic framework of 115 type strains and 23 unclassified species. Phylogeny based on 16S rRNA suggests no integrity of the earlier classified species groups, which cannot be used as a taxonomic classification. Whole genome phylogeny counter supports the non-existence of the earlier defined species groups. Genome similarity assessment suggest the existence of sixteen novel genomospecies including some of the amendments in the earlier classified species and subspecies. Highly stringent antibiotic resistance genes search resulted in widespread of fluoroquinolones resistance. Interestingly, no well-curated antibiotic resistance genes were not found in 42 genomes, including strains of probiotic importance like S. thermophilus NCTC 12958 (T) and S. salivarius NCTC 8618 (T), etc. However, presence of partial hits for resistance genes in such strains requires further experimental validation before their downstream application.

Streptococcus

phylogeny

taxonogenomic

species group

genomospecies

Streptococcus was first observed in 1868 by the German surgeon Theodor Billroth as chain-forming cocci from wounds (Wilson 1987). In 1884, Streptococcus was first used in a generic sense by Rosenbach for Streptococcus pyogenes which was isolated from suppurative lesions (Rosenbach 1884). The genus Streptococcus belongs to phylum Firmicutes, class Bacilli, order Lactobacillales, and family Streptococcaceae (Whiley and Hardie 2015). It constitutes a broad range of Gram-positive, facultatively anaerobic, spherical, or ovoid cells arranged in chains or pairs. The nomenclature of the genus has lacked international consensus (Facklam 1984). Due to which the genus had witnessed major revisions in the past with the implementation of novel techniques to understand its taxonomy. Streptococcus is misspelled with more than 64 different names similar to its true taxonomic name (https://lpsn.dsmz.de/genus/streptococcus). Genus Streptococcus was divided into three separate genera Streptococcus (sensu stricto), Enterococcus, and Lactococcus (Schleifer and Kilpper-Bälz 1984; Schleifer and Kilpper-Bälz 1987). Early taxonomy of the genus was based on biochemical, physiological, and serological characterization (Jones 1978). Lancefield serological grouping scheme (1933) is based on cell-wall polysaccharides where strains are designated as A, B, C, E, F, G, etc. (Lancefield 1933). This is extremely useful for identifying 𝛃-haemolytic streptococci (pyogenic group) from human and animal infections. However, phenotypic characteristics in earlier studies like haemolytic reaction, Lancefield grouping, or lactose fermentation cannot resolve the species of genus Streptococcus (Whiley et al. 1990). Since then, with the advancement of chemotaxonomic, whole genome DNA hybridization, and 16S rRNA gene-based taxonomy, the genus has undergone considerable taxonomic revisions (Schleifer and Kilpper-Bälz 1987; Bentley et al. 1991; Kawamura et al. 1995).

According to current standing in nomenclature, the genus Streptococcus encompasses 111 species as of October 2021 (Parte et al. 2020) (https://lpsn.dsmz.de/genus/streptococcus). The genus Streptococcus is isolated from a wide range of habitats like humans, animals, and plants. Some species such as S. thermophilus are beneficial or harmless to humans (Doron and Snydman 2015), while most streptococcal species are obligate or opportunistic pathogens like S. pyogenes, S. pneumoniae, S. equi, S. afalactiae causing pneumonia, meningitis, or endocarditis (Mitchell 2003). On the basis of 16S rRNA gene sequence and reassociation data, the constituent species of the genus Streptococcus were divided into seven “species groups” along with several ungrouped species. These seven groups are “Pyogenic”, “Bovis”, “Mutans”, “Mitis”, “Anginosus”, “Salivarius” and “Hyovaginalis” group (Bentley et al. 1991; Kawamura et al. 1995; Devriese et al. 1997; Whiley et al. 1997; Täpp et al. 2003). However, the relationship amongst these species groups is not clear and most of the species of the genus Streptococcus remain ungrouped (Richards et al. 2014; Pontigo et al. 2015) (https://lpsn.dsmz.de/genus/streptococcus). A plethora of other studies based on single genes or multilocus genes like: 16S rRNA, rpoB, gyrB, dnaJ, rnpB, recN, sodA, groEL, tuf and, rpnB have been conducted to understand its taxonomy (Kawamura et al. 1995; Täpp et al. 2003; Itoh et al. 2006; Glazunova et al. 2009; Glazunova et al. 2010). Further, genome-based investigations of the genus Streptococcus or some species groups revealed highly dynamic pan-genome (Gao et al. 2014; Richards et al. 2014; Delorme et al. 2015; Jans et al. 2015; Jensen et al. 2016; Salvadori et al. 2019). In 2018, the genus Streptococcus was reported to constitute 103 species with only 70 type strain genomes available. A study based on a limited number of species using phylogenomic and comparative genomics based on conserved sequence indels (CSI) of the genus delineated its constituent species in 2 major clades (“Mitis-Suis” and “Pyogenic-Equinus-Mutans” clades). These two major clades were further delineated into 14 other subclades (Patel and Gupta 2018). However, the analysis to redefine the species groups of the genus Streptococcus were not described in taxonomic terms. Taxonomic investigations using deep phylo-taxonogenomics (DEEPT) including genomic resources of all the representatives could assign major reconciliations in the genus Mycobacterium and Xanthomonas (Kumar et al. 2019; Bansal et al. 2021; Meehan et al. 2021). Such DEEPT investigation is required in the genus Streptococcus for a robust taxonomic framework.

Over the past two centuries, genus Streptococcus has significantly expanded with 27 novel species in past five years (https://lpsn.dsmz.de/genus/streptococcus). Currently, genus Streptococcus includes around 170 taxa (including validly published, synonyms, and not validly published species). With this unprecedented level of diversity of the species, its taxonomy becomes cumbersome. Previously attempts to understand the taxonomy of the genus Streptococcus were primarily aimed to identify the species groups and not the species. Such investigation was often based on the low resolution or limited sequence information to designate species groups based on certain physicochemical features such as the ability to haemolytic process (Brown 1920) etc. Correct taxonomic assessment can be helpful in the screening and characterization of potential probiotic strains from their pathogenic counterparts. Owing to the importance of strains of Streptococcus in dairy, healthcare, and industry, understanding their true taxonomy based on the latest robust methods is a must.

The present classification of the genus is confusing with several species, subspecies, and synonyms, which are in utmost need of a robust taxonomy. The current advances in the next-generation sequencing technologies have enabled us with generation of several genomes of the type or representative strains. Aim of the current study is to provide a robust phylo-taxono-genomic framework to the genus Streptococcus based on the gold standards for the modern taxonomy, i.e., average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) and core genome-based phylogeny (Chun et al. 2018). In addition to the type strains of the species, we have also included 23 strains with high-quality genomes which were not classified at the species level. Such large-scale genomic investigation can be helpful in biosafety assessment of the strains of genus Streptococcus. Moreover, the present study also suggests that species groups in the genus cannot be used as an absolute parameter for its taxonomy and phylogeny. Moreover, species groups cannot be used for identification and characterization of any new species of the genus. Presence of several species such as S. pneumoniae, S. pyogenes, S. agalactiae raises concern over the beneficial effect of the strain of genus Streptococcus such as S. thermophilus and S. salivarius for human (Kaci et al. 2014; Spellerberg and Brandt 2015; Alexandraki et al. 2019). Antibiotic resistance profile estimation will be crucial to understand the resistance profile of the constituent species.

Procurement of genomes from the public repository and their quality check

All the available genomes of the type species, undefined strains of genus Streptococcus, and representative of the species were procured from the NCBI genome (https://www.ncbi.nlm.nih.gov/genome/?term=Streptococcus) and EZBioCloud (https://www.ezbiocloud.net/taxonomy?tn=Streptococcus&depth=2). 16S rRNA integrity was assessed using ContEst16S (Lee et al. 2017) (https://www.ezbiocloud.net/tools/contest16s). Further, CheckM was performed for quality assessment of the genomes. Genomes showing more than 95% completeness and less than 5% contamination were considered for the analysis in the present study. Altogether, 138 high-quality genomes were used in the study from genus Streptococcus. Lactococcus lactis ATCC-19435 was used as an outgroup in the study. All the genomes were annotated using prokka v1.12 (Seemann 2014).

Phylogenomics of the genus Streptococcus

16S rRNA gene sequences were obtained for all the type strains (including species and subspecies) of genus Streptococcus (https://lpsn.dsmz.de/genus/streptococcus). A maximum likelihood (ML) based phylogenetic was constructed using MEGA v7 (Kumar et al. 2016) with a bootstrap replication of 500. This phylogenetic tree was rooted with Lactococcus lactis ATCC 19435 (T). To construct an accurate core genome phylogeny of genus Streptococcus, PEPPAN was implemented with default settings. PEPPAN implements single representative gene identification and alignment for construction of phylogeny (Zhou et al. 2020). Phylogenetic tree was annotated using iTOL (https://itol.embl.de/) for better visualization (Letunic and Bork 2019).

Taxonogenomic analysis of the genus Streptococcus

Several genome similarity assessment tools such as OrthoANI, GGDC fastANI was implemented for robust inference. OrthoANI v1.2 (Yoon et al. 2017) was implemented with a cut-off of 96% for species delineation. Digital DNA-DNA hybridization (dDDH) was estimated with a webserver of the genome-to-genome distance calculator 2.1 GGDC (http://ggdc.dsmz.de/ggdc.php) with a species delineation of 70% (Auch et al. 2010). dDDH was calculated against all the identified genomospecies across all the genome (n = 139). Heatmap for orthoANI, dDDH and was generated using GENE-E (https://software.broadinstitute.org/GENE-E/).

Resistome estimation using RGI module of CARD

RGI module (Alcock et al. 2020) (https://github.com/arpcard/rgi) of the Comprehensive Antibiotic Resistance Database (CARD, v5.2.0) was implemented for identification of antibiotic resistance gene among all the genomes in the study (n = 138). A heatmap was generated using the heatmap module of the RGI including a perfect and strict hit. Also, to obtain distant homologs of antibiotic resistance genes, a loose algorithm was used with a cut-off of > 30% and > 70% for best identity and length, respectively.

Genomic features of strains used in the study

A total of 138 high-quality genomes were used in the present study with an average genome size ranging from 1.5 mb to 2.8 mb and 33–46% GC. A descriptive table of all the species, representative and undefined species of genus Streptococcus with their genomic attributes including numbers of CDS, genome size, GC content, isolation source etc. is summarized in supplementary table 1. Genomes of species such as S. danieliae, S. symci, S. devriesei were removed from the study because of the non-permissible range of contamination and completeness or the absence of 16S rRNA gene in their genome.

Phylogenomics resolved intermingled clades and species groups within the genus Streptococcus

16S rRNA-based phylogeny of all the type strains, representative and undefined species of genus Streptococcus suggested several intermingling phylogenetic positioning of earlier defined species groups (Fig. 1). We could identify several phylogenetic positioning such as, some of the type strains of Mitis-Suis clade such as S. suis, S. azizii, S. marmotae, S. himanayensis, S. cuniculi, S. varani, S. minor, S. ovis and S. merionis shares phylogeny with Pyogenes-Equinus-Mutans clades. Likewise, the S. downii of the Pyogenes-Equinus-Mutans clade is within the Mitis-Suis clade. These evidences clearly indicate that 16S rRNA-based phylogeny could not provide sufficient evidence for supporting the species groups among genus Streptococcus.

Moreover, the current phylogeny of the genus Streptococcus is based on MLST or CSI, including a maximum of 70 species. In the present analysis, inclusion of 115 type strains along with 23 unclassified strains at whole genome level provides us robust phylogenetic grouping of the genus Streptococcus (Fig. 2). We obtained two phylogroups in accordance with the earlier clades of genus Streptococcus i.e., Mitis-Suis and Pyogenes-Equinus-Mutans. However, there were some exceptions such as: S. acidominimus, S. porci and S. plurextorum belong to Mitis-Suis clade and S. pharyngis belong to Pyogenes-Equinus-Mutans clade contrary to their previous phylogeny. Additionally, we could also designate 23 unclassified strains of genus Streptococcus to their respective clades (14 in Mitis-Suis clade and 9 in Pyogenes-Equinus-Mutans clade).

Overall, several ambiguities in subclade level grouping could not be resolved by 16S rRNA or whole genome-based phylogeny. For instance, members of subclade Halotolerans (S. acidominimus, S. hyovaginalis, S. pluranimalium, S. halotolerans and S. thoraltensis) and subclade Entericus (S. entericus, S. marimammalium) of Pyogenes-Equinus-Mutans clade had intermingled positions at both the levels of phylogeny. Interestingly, out of all these seven members of Pyogenes-Equinus-Mutans clade, S. acidominimus now belongs to Mitis-Suis clade. To further examine these ambiguities in the genus Streptococcus, we performed genome similarity assessment.

Taxonogenomics suggest the presence of several genomospecies and subspecies reclassification

The current taxonomy of the genus Streptococcus is majorly based on biochemical and physiological methods. In the study, implementation of several genome similarity assessment methods helped in identification of several novel genomospecies and in truly demarcating the species and subspecies of genus Streptococcus (Fig. 3, Data sheet 1). Genomotaxonomy revealed twelve novel genomospecies amongst the unclassified species: S. sp HKU75 (GS1), S. sp. zq-86 (GS2), S. sp. KS 6 (GS3), S. sp. LPB0220 (GS4), S. sp A12 (GS5), S. sp oral taxon 431 F0610 5-114 (GS6), S. sp oral taxon 061 F0704 (GS7), S. sp. 116-D4 (GS8), S. sp oral taxon 064 W10853 (GS9), S. sp NPS 308 (GS11) and S. sp. 1643 (GS12) and S. sp CNU G3, S. sp CNU G2, S. sp CNU 77 − 61 (GS15). Further, three of the subspecies of S. oralis (S. oralis subsp oralis NCTC 11427 (T), S. oralis subsp dentisani CECT 7747 (T) and S. oralis subsp tigurinus AZ 3a (T)) and S. equi subsp ruminatorum CECT 5772 (T) were found to be novel species and designated as GS10, GS13, GS14, and GS16.

Further, the species status of some of the species of genus Streptococcus needs to be investigated in details like: S. ilei I-G2 (T) and S. koreensis JS71 (T) (ANI: 96.32; dDDH: 68.8); S. ratti FA-1 (T) and S. ursoris DSM 22768 (T) (ANI: 98.6; dDDH: 88) and S. bovis ATCC-33317 (T) with S. equinus NCTC 12969 (T) (ANI: 96.89; dDDH: 72.7). In the present study, we could also assign nine unclassified species to already known species of the genus Streptococcus. Like, S. sp DAT741 to S. ruminantium GUT-187 (T) (ANI:99; dDDH: 90.4), S. sp. NSJ-72 to S. constellatus subsp pharyngis CCUG-46377 (T) (ANI:97.73; dDDH: 78.3), S. sp ZB199 to S. parasanguinis ATCC 15912 (T) (ANI: 96.78; dDDH: 70.8), S. sp FDAARGOS 192 to S. salivarius NCTC 8618 (T) (ANI: 96.08; dDDH: 66.5); S. sp NCTC 11567 to S. dysgalactiae subsp dysgalactiae NCTC 13731 (T) (ANI:96.02; dDDH: 67.4); S. sp FDAARGOS 522, S. sp group B FDAARGOS 229, S. sp FDAARGOS 521, S. sp FDAARGOS 520 belong to S. agalactiae NCTC 8181 (T) (ANI value in the range of 98.7% − 99.8% and dDDH value in the range of 89.6% − 98.6%).

In the present study, we have obtained 16 novel genomospecies among the genus Streptococcus. These genomospecies were classified earlier as either subspecies of already reported species or as unclassified species. Further, merger of a few already defined species such as S. ilei and S. koreensis, S. bovis, S. equinus and S. equinus, S. ratti and S. ursoris.

Genus-wide resistome analysis

Infection caused by several species of genus Streptococcus, such as S. pneumoniae, S. pyogenes etc. are the major cause of community-acquired respiratory infections worldwide (Appelbaum 2002). Antimicrobial resistance in the bacterial population raises concern over the application of microbes in industry and agriculture. Resistome analysis of the genus Streptococcus resulted in several classes of drug resistance genes using high stringent cut-offs (see methods) (Fig. 2). Resistome gene profile included: AAC(6')-Ie-APH(2'')-Ia, ANT(6)-Ia, APH(3')-IIIa and aad(6) for aminoglycosides; dfrF for diaminopyrimidines; patA, patB and pmrA for fluoroquinolones; ErmB, RlmA(II), cfr(D), lmrP, lnuA, lnuB, lnuC, lsaC, lsaE and mel for lincosamides; ErmB, RlmA(II), lmrP, lsaC, lsaE, mel for macrolides; SAT-4 for nucleosides; cfr(D), lsaC, lsaE and mel for oxazolidinones; S. agalactiae mprF for peptidic antibiotic; Enterococcus faecalis chloramphenicol acetyltransferase; Lactobacillus reuteri cat-TC, catQ, cfr(D), lsaC, lsaE and mel for phenicols; lsaC, lsaE and mel for pleuromutilin antibiotic; ErmB, cfr(D), lmrP, lsaC, lsaE and mel for streptogramin antibiotic and lmrP, lsaC, lsaE, mel, tet(L), tet(W/N/W), tetA(46), tetM and tetO for tetracycline antibiotic (Data sheet 2). Most of these resistance genes were present in S. porci DSM 23759, S. gallolyticus subsp pasteurianus NCTC 13784, S. hyovaginalis DSM 12219, S. parasuis H35, S. plurextorum DSM 22810, S. orisratti DSM 15617, S. pluranimalium TH11417, S. sp FDAARGOS 522 and S. vaginalis P1L01 (Fig. 2). Further, 75 out of 138 genomes were harbouring fluoroquinolone resistance genes (patA and patB). Interestingly, well-curated antibiotic resistance genes were not found in 42 genomes in the present study. These genomes were majorly present in Pyogenes-Equinus-Mutans clade in phylogroup containing strains of probiotic importance like: S. thermophilus NCTC 12958 (T) and S. salivarius NCTC 8618 (T) etc. Further, Mitis-Suis clade also had 14 genomes not having perfect hits for resistance genes (Fig. 2).

However, low-stringent cut-offs (see methods) resulted in a highly enriched resistome profile for all the species of the genus Streptococcus (Data sheet 3). A total of 61,274 antibiotic resistance-related genes could be identified using the standalone RGI module of CARD. Out of which, 15,976 genes were efflux pump related genes. Antibiotic resistance genes related to β-lactam were detected in all the strains in large numbers (up to 178 copies). Aminoglycoside and glycopeptide antibiotics were also present in large numbers among all the strains. Few strains of genus harbour gene related to ethionamide, fluoroquinolone antibiotic; aminoglycoside antibiotic, isoniazid; triclosan, mupirocin, polyamine antibiotic, prothionamide, pyrazinamide, and sulfone antibiotic.

Present study is an attempt to understand the taxonomy and status of species group previously described based on the whole genome information. We have curated all the type strain information of the species of genus Streptococcus to perform a systematic phylo-taxonogenomic investigation. We found several taxonomic ambiguities and existence of genomospecies within genus Streptococcus. Such a genome wide investigation will be very important in understanding the nature of constituent species including clinical, commensal and industrial important strains. Resistome profile for species of probiotic or industrial importance like S. thermophilus NCTC 12958 (T) or S. salivarius NCTC 8618 (T) etc was very diverse or partially matched. These antibiotic resistance gene hits at low stringent cutoffs needs to be further experimentally verified. This will enable the research and industrial community to truly demarcate strains for their downstream applications.

CSI: conserved sequence indels; ANI: Average Nucleotide Identity; dDDH: digital DNA-DNA hybridization; ML: maximum likelihood; CARD: Comprehensive Antibiotic Resistance Database; RGI: Resistance gene identifier; GS: genomospecies

Funding Information

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Conflict of Interest

All the authors declare no conflict of interest.

Author Contributions

SK and KB have performed all the investigation and drafted the manuscript. SK, KB. SKS and SK have conceived the manuscript.

Ethics approval

This is an observational study which does not require any ethical approval.

Acknowledgement

All the authors convey their regards for support from Ms. Anu Singh, research scholar at CSIR-Institute of Microbial Technology, Chandigarh for her valuable discussion and support. We also convey a special thanks to Dr. Prabhu Patil for their immense support and guidance through the whole study.

Data Availability

All the data sheets generated in the manuscript has been provided with the figshare link: (https://figshare.com/articles/dataset/Data_sheets_for_the_study/19312352). Description of each data sheets can be found as fellow:

Data sheet 1: Digital DNA-DNA hybridization (dDDH) values of the strains with taxonomic ambiguities in the genus Streptococcus.

Data sheet 2: Antibiotic resistance drug classes identified by RGI module of CARD with high stringent cutoffs.

Data sheet 3: All the identified antibiotic resistance gene with identity and coverage cutoff greater than 30% and 70% respectively.

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Phylo-taxonogenomic reconciliations of genus Streptococcus reveals the existence of sixteen novel genomospecies

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