In collaboration with the CDC’s Streptococcus Laboratory, we report here the whole-genome sequences of seven Streptococcus agalactiae bacteria isolated from laboratory-reared Long-Evans rats. Four of the S. agalactiae isolates were associated with morbidity accompanied by endocarditis, metritis, and fatal septicemia, providing an opportunity for comparative genomic analysis of this opportunistic pathogen.United States. National Institutes of Health (T32-OD010978)United States. National Institutes of Health (P30-ES002109

Bakthavatchalu, Vasudevan

Bodi Winn, Caroline M

Dzink-Fox, J.

Feng, Yan

Fox, James G

Shen, Zeli

Genome Announcements

PubMed

ABSTRACT
          
            In collaboration with the CDC’s
            Streptococcus
            Laboratory, we report here the whole-genome sequences of seven
            Streptococcus agalactiae
            bacteria isolated from laboratory-reared Long-Evans rats. Four of the
            S. agalactiae
            isolates were associated with morbidity accompanied by endocarditis, metritis, and fatal septicemia, providing an opportunity for comparative genomic analysis of this opportunistic pathogen.
          </jats:p

C. Bodi Winn

J. Dzink-Fox

Y. Feng

Z. Shen

V. Bakthavatchalu

J. G. Fox

Crossref

Whole-Genome Sequences and Classification of Streptococcus agalactiae Strains Isolated from Laboratory-Reared Long-Evans Rats (Rattus norvegicus)

DSpace@MIT

Whole-Genome Sequences andClassiﬁcation of Streptococcus agalactiaeStrains Isolated from Laboratory-RearedLong-Evans Rats (Rattus norvegicus)C. Bodi Winn, J. Dzink-Fox, Y. Feng, Z. Shen, V. Bakthavatchalu, J. G. FoxDivision of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts, USAABSTRACT In collaboration with the CDC’s Streptococcus Laboratory, we report herethe whole-genome sequences of seven Streptococcus agalactiae bacteria isolatedfrom laboratory-reared Long-Evans rats. Four of the S. agalactiae isolates were asso-ciated with morbidity accompanied by endocarditis, metritis, and fatal septicemia,providing an opportunity for comparative genomic analysis of this opportunisticpathogen.Streptococcus agalactiae, or group B streptococcus (GBS), is a Gram-positive oppor-tunistic pathogen that commonly inhabits the gastrointestinal and urogenital tractof humans. It is the leading cause of neonatal septicemia and meningitis, and it is alsoknown to cause disease in animals, including meningoencephalitis in ﬁsh, mastitis incattle, and invasive infection in laboratory rodents (1–6). There is high suspicion thatGBS is a zoonotic pathogen, and despite close genotypic relationships observedbetween different species’ isolates, direct transmission has not been reported (7–9). Inthis report, we announce the whole-genome sequences of seven GBS isolates obtainedfrom a colony of laboratory-reared Long-Evans rats that experienced two cases ofpolymicrobial sepsis associated with GBS infection in two gravid female rats; twoisolates were obtained from resected cardiac tissue and blood in the ﬁrst case, and twoisolates were obtained from cardiac tissue and fetus from the second case. An addi-tional three asymptomatic male rats’ GBS isolates were cultured from the nares. Isolateswere 16S rRNA sequenced and submitted to the CDC’s Streptococcus Laboratory forwhole-genome sequencing.The CDC’s Streptococcus Laboratory tracks cases of GBS invasive disease throughtheir Active Bacterial Core surveillance (ABCs). After receipt of isolates, genomic DNA forshort-read whole-genome sequencing was extracted manually using a modiﬁedQIAamp DNA minikit protocol (Qiagen). Nucleic acid concentration was quantiﬁed byan Invitrogen Qubit assay, and samples were sheared using a Covaris M220 ultrasoni-cator programmed to generate 500-bp fragments. Libraries were constructed usingTruSeq DNA PCR-free high-throughput (HT) library preparation kit with 96 dual indexesand quantiﬁed by a Kapa quantitative PCR (qPCR) library quantiﬁcation method.Genomes were sequenced by Illumina NGS technology using MiSeq version 2 500-cyclekit. Genomes were annotated using Rapid Annotations using Technology toolkit(RASTtk) (10, 11) (http://rast.nmpdr.org/rast.cgi).Strains underwent the following analyses: (i) multilocus sequence typing (MLST;http://pubmlst.org/sagalactiae) (12), (ii) serotyping (13), (iii) detection of resistancegenes with ResFinder (14) and the Comprehensive Antibiotic Resistance Database(CARD) (15), (iv) virulence factors (VFDB; http://www.mgc.ac.cn/VFs/) (16), and (v)antimicrobial susceptibility testing by a microdilution broth method.Received 26 October 2016 Accepted 1November 2016 Published 5 January 2017Citation Bodi Winn C, Dzink-Fox J, Feng Y,Shen Z, Bakthavatchalu V, Fox JG. 2017. Whole-genome sequences and classiﬁcation ofStreptococcus agalactiae strains isolated fromlaboratory-reared Long-Evans rats (Rattusnorvegicus). Genome Announc 5:e01435-16.https://doi.org/10.1128/genomeA.01435-16.Copyright © 2017 Bodi Winn et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to J. G. Fox,jgfox@mit.edu.PROKARYOTEScrossmVolume 5 Issue 1 e01435-16 genomea.asm.org 1 on May 19, 2017 by MASS INST OF TECHNOLOGYhttp://genomea.asm.org/Downloaded from MLST analysis and serotyping assigned all GBS strains except strain 195-16-B-RAT toserotype Ib, sequence type 12 (ST12) (13, 17). Strain 195-16-B-RAT is serotype V, ST1.Both serotype-sequence types are well-represented among human invasive isolatesdocumented in the 2015 ABCs (unpublished data). Isolates were susceptible to ampi-cillin (0.25 g/ml), ceftriaxone (0.5 g/ml), erythromycin (0.25 g/ml), and clin-damycin (0.25 g/ml) but resistant to tetracycline (8 g/ml). Strains were interme-diate resistant to enroﬂoxacin (0.5 g/ml), a commonly used ﬂuoroquinolone inveterinary medicine (18, 19). Strain 195-16-B-RAT had a mutation detected in the ermAgene, denoting resistance to erythromycin. This may be due to inducible expression ofthe resistance-causing methylase enzyme, which leads to different phenotypes (20). Allisolates had tet(M), associated with tetracycline resistance. The virulence factors iden-tiﬁed were -hemolysin/cytolysin (cylE), CAMP factor (cfa/cfb), hyaluronate lyase (hylB),C5a peptidase (scpB), pili (pilA, pilB, pilC), sortases (srtC3, srtC4), and Fg-binding surfaceprotein A and B (fbsA and fbsB).Accession number(s). The whole-genome sequences of the seven S. agalactiaestrains have been deposited at DDBL/EMBL/GenBank under the accession numbersprovided in Table 1.ACKNOWLEDGMENTSWe thank Bernard Beall, Sopio Chochua, and Benjamin Metcalf at the CDC’s Strep-tococcus Laboratory for whole-genome sequencing, technical support, and collabora-tion. We thank Anthony Mannion for technical guidance. We thank Alyssa Pappa for herexcellent assistance. We also thank Michael Esmail, Lauren Richey, and Scott Perkins fortheir contributions and support.This work was supported in part by NIH grants T32-OD010978 (J.G.F.) and P30-ES002109 (J.G.F.).REFERENCES1. Verani JR, McGee L, Schrag SJ. 2010. Prevention of perinatal group Bstreptococcal disease—revised guidelines from CDC, 2010. MMWR Re-comm Rep 59:1–32.2. Liu G, Zhang W, Lu C. 2012. Complete genome sequence of Streptococ-cus agalactiae GD201008 -001, isolated in China from tilapia with me-ningoencephalitis. J Bacteriol 194:6653. https://doi.org/10.1128/JB.01788-12.3. Schenkman DI, Rahija RJ, Klingenberger KL, Elliott JA, Richter CB. 1994.Outbreak of group B streptococcal meningoencephalitis in athymicmice. Lab Anim Sci 44:639–641.4. Keefe GP. 1997. Streptococcus agalactiae mastitis: a review. Can Vet J38:429–437.5. Shuster KA, Hish GA, Selles LA, Chowdhury MA, Wiggins RC, Dysko RC,Bergin IL. 2013. Naturally occurring disseminated group B streptococcusinfections in postnatal rats. Comp Med 63:55–61.6. Geistfeld JG, Weisbroth SH, Jansen EA, Kumpfmiller D. 1998. Epizootic ofgroup B Streptococcus agalactiae serotype V in DBA/2 mice. Lab Anim Sci48:29–33.7. Manning SD, Springman AC, Million AD, Milton NR, McNamara SE,Somsel PA, Bartlett P, Davies HD. 2010. Association of group Bstreptococcus colonization and bovine exposure: A prospectivecross-sectional cohort study. PLoS One 5:e8795. https://doi.org/10.1371/journal.pone.0008795.8. Delannoy CMJ, Crumlish M, Fontaine MC, Pollock J, Foster G, DagleishMP, Turnbull JF, Zadoks RN. 2013. Human Streptococcus agalactiaestrains in aquatic mammals and ﬁsh. BMC Microbiol 13:41. https://doi.org/10.1186/1471-2180-13-41.9. Lämmler C, Abdulmawjood A, Weiß R. 1998. Properties of serologicalgroup B streptococci of dog, cat and monkey origin. Zentralbl Vet B45:561–566. https://doi.org/10.1111/j.1439-0450.1998.tb00827.x.10. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K,Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL,Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD,Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008.The RAST server: rapid annotations using subsystems technology. BMCGenomics 9:75. https://doi.org/10.1186/1471-2164-9-75.11. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R,Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA, Stevens R,Vonstein V, Wattam AR, Xia F. 2015. RASTtk: a modular and extensibleimplementation of the RAST algorithm for building custom annotationpipelines and annotating batches of genomes. Sci Rep 5:8365. https://doi.org/10.1038/srep08365.12. Moltó-García B, Liébana-Martos Mdel C, Cuadros-Moronta E, Rodríguez-TABLE 1 Genomic characteristics of rat Streptococcus agalactiae strainsS. agalactiaestrainGenBankaccession no.Origin of ratisolatesPresence ofclinical diseaseGenomesize (bp)No. ofcontigsFoldcoverageGCcontent (%)No. of protein-codingsequencesNo. of RNAsequences195-16-B-RAT MJHP00000000 Nares No 2,134,553 22 50 35.2 2,146 56196-16-B-RAT MJHQ00000000 Blood Yes 2,060,190 41 50 35.1 2,064 61197-16-B-RAT MJHR00000000 Cardiac lesion Yes 2,065,096 35 50 35.1 2,072 61198-16-B-RAT MJHS00000000 Nares No 2,060,782 38 50 35.1 2,078 65199-16-B-RAT MJHT00000000 Nares No 2,074,289 37 50 35.1 2,080 68200-16-B-RAT MJHU00000000 Cardiac lesion Yes 2,068,751 37 100 35.1 2,084 76201-16-B-RAT MJHV00000000 Fetus Yes 2,063,718 35 100 35.1 2,082 70Bodi Winn et al.Volume 5 Issue 1 e01435-16 genomea.asm.org 2 on May 19, 2017 by MASS INST OF TECHNOLOGYhttp://genomea.asm.org/Downloaded from Granger J, Sampedro-Martínez A, Rosa-Fraile M, Gutierrez-Fernández J,Puertas-Priet A, Navarro-Marí JM. 2016. Molecular characterization andantimicrobial susceptibility of hemolytic Streptococcus agalactiae frompost-menopausal women. Maturitas 85:5–10. https://doi.org/10.1016/j.maturitas.2015.11.007.13. Sheppard AE, Vaughan A, Jones N, Turner P, Turner C, Efstratiou A, PatelD, Modernising Medical Microbiology Informatics Group, Walker AS,Berkley JA, Crook DW, Seale AC. Capsular typing method for Streptococ-cus agalactiae using whole genome sequence data. J Clin Microbiol54:1388–1390. https://doi.org/10.1128/JCM.03142-15.14. Kleinheinz KA, Joensen KG, Larsen MV. 2014. Applying the ResFinder andVirulenceFinder Web-services for easy identiﬁcation of acquired antibi-otic resistance and E. coli virulence genes in bacteriophage and pro-phage nucleotide sequences. Bacteriophage 4:e27943. https://doi.org/10.4161/bact.27943.15. McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, BhullarK, Canova MJ, De Pascale G, Ejim L, Kalan L, King AM, Koteva K, Morar M,Mulvey MR, O’Brien JS, Pawlowski AC, Piddock LJV, SpanogiannopoulosP, Sutherland AD, Tang I, Taylor PL, Thaker M, Wang W, Yan M, Yu T,Wright GD. 2013. The comprehensive antibiotic resistance database.Antimicrob Agents Chemother 57:3348–3357. https://doi.org/10.1128/AAC.00419-13.16. Chen L, Yang J, Yu J, Yao Z, Sun L, Shen Y, Jin Q. 2005. VFDB: a referencedatabase for bacterial virulence factors. Nucleic Acids Res 33:D325–D328. https://doi.org/10.1093/nar/gki008.17. Jones N, Bohnsack JF, Takahashi S, Oliver KA, Chan MS, Kunst F, GlaserP, Rusniok C, Crook DWM, Harding RM, Bisharat N, Spratt BG. 2003.Multilocus sequence typing system for group B streptococcus. J ClinMicrobiol 41:2530 –2536. https://doi.org/10.1128/JCM.41.6.2530-2536.2003.18. Clinical and Laboratory Standards Institute (CLSI). 2013. Performancestandards for antimicrobial disk and dilution susceptibility tests forbacteria isolated from animals; approved standard, 4th ed. CLSI docu-ment VET01-A4. Clinical and Laboratory Standards Institute, Wayne, PA.19. Clinical and Laboratory Standards Institute (CLSI). 2013. Performancestandards for antimicrobial susceptibility testing; 23rd informationalsupplement. CLSI document M100-S23. Clinical and Laboratory Stan-dards Institute, Wayne, PA.20. Leclercq R. 2002. Mechanisms of resistance to macrolides andlincosamides: nature of the resistance elements and their clinical impli-cations. Clin Infect Dis 34:482–492. https://doi.org/10.1086/324626.Genome AnnouncementVolume 5 Issue 1 e01435-16 genomea.asm.org 3 on May 19, 2017 by MASS INST OF TECHNOLOGYhttp://genomea.asm.org/Downloaded from 

Whole-Genome Sequences and Classification of

http://dx.doi.org/10.1128/genomeA.01435-16

Whole-Genome Sequences and Classification of

Abstract

Similar works

Full text

Available Versions

Crossref

DSpace@MIT