Isolation of human GBS CC67 strains with high adhesion ability to HBMECs
Authors: J. Hu, L. Li, R. Wang, T. Huang, Y. Huang, Y. Liu, X. Yang, M. Chen
Journal: Clinical Microbiology and Infection (2019)

To the editor, The Group B Streptococcus (GBS) strain is a very important pathogen in both humans and bovines [[1]Sorensen U.B.S. Poulsen K. Ghezzo C. Margarit I. Kilian M. Emergence and global dissemination of host-specific Streptococcus agalactiae clones.Mbio. 2010; 1 (00178-10)Crossref PubMed Scopus (113) Google Scholar]. Multilocus sequence typing (MLST) analyses of GBS from different countries and animals show that most human carriage and pathogenic isolates are clustered into five major clonal complexes (CCs), namely CC1, CC10, CC17, CC19, and CC23, while the majority of bovine isolates belong to CC67 [[2]Da Cunha V. Davies M.R. Douarre P.E. Rosinski-Chupin I. Margarit I. Spinali S. et al.Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline.Nat Commun. 2014; 5: 4544Crossref PubMed Scopus (148) Google Scholar]. Whether bovine GBS can cross-host infect humans has been very controversial in the past 50 years. So far, no bovine CC67 strains have been isolated and validated from human. In this study, 92 isolates from 92 patients with 19 different diseases were identified as Streptococcus agalactiae in Guangxi China (Table S1 and Fig. S1). Among them are three special strains: NNA006, BSE003, and BSE004 (Fig. 1A). BSE004 belongs to sequence type (ST) 61. BSE003 is a double-locus variant (DLV) of BSE004, and NNA006 is a DLV of BSE003. NNA006 and BSE003 belong to new STs, named ST929 and ST931. Maximum likelihood trees based on both 12 housekeeping genes and whole genome sequences indicated that these three strains were clustered into bovine CC67 lineage (Fig. S2). It is reasonable to reach the conclusion that these three strains are human source GBS CC67 strains. Notably, three patients infecting the above three strains live in two cities 200 km away. It reminds us that presence of these human CC67 strains is not incidental and long-distance spatial dissemination is possible. Chromosomal replacement and gene recombination among different CCs of GBS may produce many new ST and terrible drug-resistant GBS strain. We focused on a typical cell wall-anchored surface protein, Gbs2018. This protein was clustered into four major clusters, designated, A, B, C, and D (Fig. S3). To explore the structural diversity of this protein, ten fragments were roughly defined according to sequence similarity, namely F1–F10 (Fig. S3). Gbs2018 proteins of NNA006 and BSE003 were clustered into lineage D (Fig. S3). However, they acquired a lineage-C-specific fragment F9 (Figs. 1B and Fig. S4) and shared higher structural similarity to Gbs2018 proteins in lineage C rather than D (Fig. S5). Lineage C Gbs2018 is also called hypervirulent GBS adhesin gene (HvgA) and is CC17 specific [[3]Lamy M.C. Dramsi S. Billoet A. Reglier-Poupet H. Tazi A. Raymond J. et al.Rapid detection of the “highly virulent” group B Streptococcus ST-17 clone.Microbe. Infect. 2006; 8: 1714-1722Crossref PubMed Scopus (98) Google Scholar]. It mediates GBS colonization in neonatal intestine and crossing the intestinal and blood–brain barriers, leading to meningitis [[4]Tazi A. Disson O. Bellais S. Bouaboud A. Dmytruk N. Dramsi S. et al.The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates.J Exp Med. 2010; 207: 2313-2322Crossref PubMed Scopus (194) Google Scholar]. So, we hypothesized that Gbs2018 in NNA006 and BSE003 might mimic the function of HvgA proteins, thus contributing to the adhesion and colonization ability of these two isolates. We selected three human CC17 strains as positive control and two popular bovine strains ST61 and ST103 as negative control. Notably, NNA006 and BSE003, which acquire fragment F9 from CC17 GBS HvgA gene, have hyper-adhesion ability to human brain microvascular endothelial cells (HBMECs). To make matters worse, the pathogenicity of the two strains seems to exceed the notorious CC17 strains. By contrast, strain BSE004 does not obtain F9 fragment and has very low adhesion ability to HBMECs (Fig. 1C). After that, we made some analysis on the origin of these new strains. The following evidence suggested that they might arise from gene recombination of bovine CC67 and human CC17 strains. The following are new strains: (1) clustered with bovine CC67 strains; (2) harboured CC17-specific fragment F9; (3) had heterozygous terminal repeat in CRISPR system [[5]Shipman S.L. Nivala J. Macklis J.D. Church G.M. Molecular recordings by directed CRISPR spacer acquisition.Science (New York, NY). 2016; 353 (aaf1175)Crossref Scopus (123) Google Scholar] (Table S2); (4) had three CC17 DNA fragments (Fig. S6). Strangely, no CC17 strains were isolated from patient P1, P2, and P3. It seemed to be that the recombination occurred in an unknown host rather than in these three patients. We made detailed analysis to find trace of the unknown host: (1) new strains acquired three CC17 fragments that encoded 17 proteins. Fourteen of them are bacterial surface membrane proteins and can help CC17 adapt to human environment (please see supplementary material); (2) the Lac.2 operon presented in all of these bovine CC67 strains, but not in CC17 strains (Table S3). It seemed that the Lac.2 operon helped GBS stains adapt to the bovine rather than the human environment. Although new strains grouped with bovine CC67 strains, none of them harboured the Lac.2 operon (Table S3). Considering the above analysis, we proposed two models to explain the recombination and transmission event of the human CC67 strains (Fig. S7). We think the first model is more close to the real process: (1) bovine CC67 strains infect human CC17 carriers and then reassort with CC17 strains, generating the recombinant CC67 strains; (2) genomes of recombinant CC67 strains interact with the human environment, resulting variations in traits, such as losing the Lac.2 operon or acquiring surface membrane genes. Over a long period of time, they finally become human adaptive GBS strains, like BSE004; (3) once the F9 fragment from CC17 strains has been acquired, human adaptive CC67 strains become hypervirulent CC67 strains, like NNA006 and BSE003. Next, we will conduct an in-depth research on the mechanism of CC67 interspecies transmission to provide a theoretical basis for effective prevention and control of the disease. We declare no competing interests. National Natural Science Foundation of China (Grant no. 31460695). Guangxi innovation driven development special funds (Grant no. AA17204081-3). We thank Li Ye and Xiaohan Chen for revising the manuscript. The following is the Supplementary data to this article: Download .docx (9.77 MB) Help with docx files Multimedia component 1