New Insights on Streptococcus dysgalactiae subsp. dysgalactiae Isolates

Abstract

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been considered a strict animal pathogen. Nevertheless, the recent reports of human infections suggest a niche expansion for this subspecies, which may be a consequence of the virulence gene acquisition that increases its pathogenicity. Previous studies reported the presence of virulence genes of Streptococcus pyogenes phages among bovine SDSD (collected in 2002-2003); however, the identity of these mobile genetic elements remains to be clarified. Thus, this study aimed to characterize the SDSD isolates collected in 2011-2013 and compare them with SDSD isolates collected in 2002-2003 and pyogenic streptococcus genomes available at the National Center for Biotechnology Information (NCBI) database, including human SDSD and S. dysgalactiae subsp. equisimilis (SDSE) strains to track temporal shifts on bovine SDSD genotypes. The very close genetic relationships between humans SDSD and SDSE were evident from the analysis of housekeeping genes, while bovine SDSD isolates seem more divergent. The results showed that all bovine SDSD harbor Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas IIA system. The widespread presence of this system among bovine SDSD isolates, high conservation of repeat sequences, and the polymorphism observed in spacer can be considered indicators of the system activity. Overall, comparative analysis shows that bovine SDSD isolates carry speK, speC, speL, speM, spd1, and sdn virulence genes of S. pyogenes prophages. Our data suggest that these genes are maintained over time and seem to be exclusively a property of bovine SDSD strains. Although the bovine SDSD genomes characterized in the present study were not sequenced, the data set, including the high homology of superantigens (SAgs) genes between bovine SDSD and S. pyogenes strains, may indicate that events of horizontal genetic transfer occurred before habitat separation. All bovine SDSD isolates were negative for genes of operon encoding streptolysin S, except for sagA gene, while the presence of this operon was detected in all SDSE and human SDSD strains. The data set of this study suggests that the separation between the subspecies "dysgalactiae" and "equisimilis" should be reconsidered. However, a study including the most comprehensive collection of strains from different environments would be required for definitive conclusions regarding the two taxa.

Keywords: CRISPR typing; Streptococcus dysgalactiae subsp. dysgalactiae; phage genes; phylogenetic relationships; virulence genes.

FIGURE 1

Phylogenetic analysis based on concatenated sequences of seven housekeeping genes (gki, gtr, murI, mutS, recP, xpt, and atoB) showing the position of the 15 bovine SDSD isolates and sequences of strains available in the National Center for Biotechnology Information (NCBI) database, including SDSD strains (n = 12), SDSE strains (n = 15), Streptococcus pyogenes strains (n = 11), Streptococcus canis strains (n = 3). VSD, Vet S. dysgalactiae subsp. dysgalactiae. Bovine SDSD isolates: ATCC 27957; NCTC13731; NCTC4669; NCTC4670 and VSDs. Human SDSD strains: DB31752-13; DB49998-05; DB60705-15, and DB53993-17. Fish SDSD strain: SDSD Kdys0611. SDSE, S. dysgalactiae subsp. equisimilis. The phylogenetic relationships were inferred using the minimum evolution method, and phylogenetic distances were estimated using the maximum composite likelihood method using the MEGA (Kumar et al., 2018). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

FIGURE 2

Distribution (%) of group A Streptococcus pyogenes virulence gene among Streptococcus dysgalactiae subsp. dysgalactiae collections isolate in 2002–2003 and 2011–2013. speC, speK, speL, speM: encode streptococcal pyrogenic exotoxins; spd1: DNase; sdn: streptodornase (DNase). The statistical significance of the data was determined by a chi-square test (χ²), where a probability value (p) ≤ 0.05 was considered as statistically significant.

FIGURE 3

Gene cluster organization of streptolysin S (SLS) operon and precursor peptide sequences of SLS cytolysins (SagA). (A) Organization of the SLS operon in Streptococcus dysgalactiae subsp. equisimilis (SDSE), human and fish S. dysgalactiae subsp. dysgalactiae (SDSD), Streptococcus pyogenes, and Streptococcus canis. The operon encoding SLS includes the prepropeptide structural gene (sagA), followed by eight genes responsible for the conversion of SagA into SLS (sagBCD), transport across the membrane (sagFGHI), and accountable for leader cleavage (sagE) (Datta et al., 2005). (B) Organization of the genomic region flanking sagA in bovine SDSD isolates. (C) Alignment of SagA peptide, the precursor of SLS. SLS core region possesses a highly conserved N-terminus, while the C-terminus is more variable. The putative leader peptide cleavage site is shown. Red rectangle, minimal core region required for hemolytic activity of SLS in S. pyogenes. STAB-Vida performed sequencing of sagA. Deduced amino acid sequences from this bovine allele were compared with sequences from the National Center for Biotechnology Information (NCBI) database and were analyzed with the CLC-bio Main Workbench sequence alignment tool (QIAGEN, Netherlands).

FIGURE 4

(A) Percentages of antimicrobial resistance among bovine Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) isolates from the present study against erythromycin (ERY), gentamicin (CN), pirlimycin (PRL), streptomycin (S), and tetracycline (TET). Resistance was not observed against amoxicillin–clavulanic acid (AMC), cefazolin (KZ), chloramphenicol (CHL), penicillin (P), rifaximin (RAX), and vancomycin (VA). (B) Distribution resistance genotypes among bovine SDSD: ermA and ermB: macrolide resistance genes; linB: lincosamide resistance gene; tetK, tetM, and tetO: resistance to tetracycline genes. The statistical significance of the data was determined by a chi-square test (χ²), where a probability value (p) ≤ 0.05 was considered as statistically significant.

FIGURE 5

Dendrogram based on spacer CRISPR IIA cluster of Streptococcus dysgalactiae subsp. dysgalactiae isolates of mastitis in cattle collected in 2002–2003 (Rato et al., 2010, 2011, 2013) (VSD1 to VSD19—collection I) and 2011–2013 (VSD20 to VSD56—collection II). Streptococcus pyogenes phages encode genes speC, speK, speL, speM, spd1, and sdn. The dendrogram was obtained by means of Dice coefficient and the agglomerative clustering of the unweighted pair group method with arithmetic mean (UPGMA).