Molecular Characteristics of IS 1216 Carrying Multidrug Resistance Gene Cluster in Serotype III/Sequence Type 19 Group B Streptococcus

Abstract

Streptococcus agalactiae is the leading cause of meningitis in newborns and a significant cause of invasive diseases in pregnant women and adults with underlying diseases. Antibiotic resistance against erythromycin and clindamycin in group B streptococcus (GBS) isolates has been increasing worldwide. GBS expresses the Srr1 and Srr2 proteins, which have important roles in bacterial infection. They have been investigated as novel vaccine candidates against GBS infection, with promising results. But a recent study detected non-srr1/2-expressing clinical isolates belonging to serotype III. Thus, we aimed to analyze the genotypes of non-srr1/2 GBS clinical isolates collected between 2013 and 2016 in South Korea. Forty-one (13.4%) of the 305 serotype III isolates were identified as non-srr1/2 strains, including sequence type 19 (ST19) (n = 16) and ST27 (n = 18) strains. The results of the comparative genomic analysis of the ST19/serotype III/non-srr1/2 strains further revealed four unique gene clusters. Site 4 in the srr1 gene locus was replaced by an lsa(E)-lnu(B)-aadK-aac-aph-aadE-carrying multidrug-resistant gene cluster flanked by two IS1216 transposases with 99% homology to the enterococcal plasmid pKUB3007-1. Despite the Srr1 and Srr2 deficiencies, which resulted in reduced fibrinogen binding, the adherence of non-srr1/2 strains to endothelial and epithelial cells was comparable to that of Srr1- or Srr2-expressing strains. Moreover, their virulence in mouse models of meningitis was not significantly affected. Furthermore, additional adhesin-encoding genes, including a gene encoding a BspA-like protein, which may contribute to colonization by non-srr1/2 strains, were identified via whole-genome analysis. Thus, our study provides important findings that can aid in the development of vaccines and antibiotics against GBS. IMPORTANCE Most previously isolated group B streptococcus (GBS) strains express either the Srr1 or Srr2 glycoprotein, which plays an important role in bacterial colonization and invasion. These glycoproteins are potential protein vaccine candidates. In this study, we first report GBS clinical isolates in which the srr1/2 gene was deleted or replaced with foreign genes. Despite Srr1/2 deficiency, in vitro adherence to mammalian cells and in vivo virulence in murine models were not affected, suggesting that the isolates might have another adherence mechanism that enhanced their virulence aside from Srr1/2-fibrinogen-mediated adherence. In addition, several non-srr1/2 isolates replaced the srr1/2 gene with the lnu(B) and lsa(E) antibiotic resistance genes flanked by IS1216, effectively causing multidrug resistance. Collectively, we believe that our study identifies the underlying genes responsible for the pathogenesis of new GBS serotype III. Furthermore, our study emphasizes the need for alternative antibiotics for patients who are allergic to β-lactams and for those who are pregnant.

Keywords: IS1216; ST19; Streptococcus agalactiae; multidrug resistance gene; srr1/2.

FIG 1

Reduced binding of non-srr1/2 isolates to immobilized fibrinogen. (A) Expression of glycoproteins Srr1 and Srr2 on the cell surface. Cell wall proteins were isolated from Srr1-expressing GBS (NEM316) and its isogenic mutant (Δsrr1), Srr2-expressing GBS (COH1) and its isogenic mutant (Δsrr2), and non-srr1/2 isolates S9968 and NSP15-73. Srr1/2 proteins separated by SDS-PAGE were detected with biotin-conjugated WGA, followed by incubation with HRP-conjugated streptavidin. (B) GBS binding to immobilized fibrinogen. Suspensions of GBS WT (NEM316, COH1, and S9968) and isogenic mutant (NEM316 Δsrr1 and COH1 Δsrr2) strains were incubated in wells pretreated with human fibrinogen (0.1 μM), followed by detection with 0.1% crystal violet. Cell binding to immobilized fibrinogen was measured as the absorbance at 595 nm. Data are presented as the means ± standard deviations. **, P < 0.005; ***, P < 0.001.

FIG 2

Genome comparison of GBS S9968 and other serotype III isolates. (A) Phylogenetic tree analysis of the S9968 strain based on ANI values obtained from whole genome sequences. The genome sequences were retrieved from the GenBank database, and ANI values were estimated using JSpecies. The phylogenetic tree was constructed using MEGA 6.0. (B) Comparative genome visualization map of S9968 with two related genome isolates, Sag158 (GenBank accession number CP019979) and H002 (accession number CP011329). GC contents and GC skew are represented on the distance scale in the inner map. Orthologous genes found in H002 (red) and Sag158 (blue) are displayed in the outer map. The double line (green) in the map indicates the unique ORFs found in S9968.

FIG 3

Schematic representation of the antibiotic resistance locus flanked by two IS1216 transposases in GBS S9968. The genetic locus flanked by two IS1216 transposases in the site 4 cluster of the S9968 strain was compared with the corresponding regions identified in GBS strains H002 and NEM316 and E. faecalis pKUB3006.

FIG 4

Comparison of adherence rates and virulences of GBS strains. (A and B) Adherence of GBS strains, NEM316 and its isogenic mutant (Δsrr1), COH1 and its isogenic mutant (Δsrr2), and S9968, to hBMECs (A) and A549 cells (B). Data are presented as the means ± standard deviations. (C) Kaplan-Meier survival curves. CD-1 male mice were injected i.v. with GBS strain NEM316 or its isogenic mutant (Δsrr1), COH1 or its isogenic mutant (Δsrr2), or S9968 (n = 10 per group). Mouse survival was monitored for 72 h. (D to F) Bacterial loads in mouse organs. At the end of the experiment, mice were euthanized, and bacterial loads in the brain (D), lung (E), and blood (F) were assessed. *, P < 0.05; **, P < 0.01; n.s, not significant.