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Weinstock, George

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Washington University School of MedicineDigital Commons@BeckerOpen Access Publications2014Whole-genome sequencing of Salmonella entericasubsp. enterica serovar Cubana strains isolated fromagricultural sourcesGeorge WeinstockWashington University School of Medicine in St. Louiset alFollow this and additional works at: http://digitalcommons.wustl.edu/open_access_pubsThis Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in OpenAccess Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact engeszer@wustl.edu.Recommended CitationWeinstock, George and et al, ,"Whole-genome sequencing of Salmonella enterica subsp. enterica serovar Cubana strains isolated fromagricultural sources." Genome Announcements.2,1. e01184-13. (2014).http://digitalcommons.wustl.edu/open_access_pubs/2159Whole-Genome Sequencing of Salmonella enterica subsp. entericaSerovar Cubana Strains Isolated from Agricultural SourcesFaiza H. Benahmed,a Gopal R. Gopinath,b Hua Wang,c Junia Jean-Gilles Beaubrun,b Christopher Grim,b Chorng-Ming Cheng,dMichael McClelland,e Sherry Ayers,a Jason Abbott,a Prerak Desai,e Jonathan G. Frye,f George Weinstock,g Thomas S. Hammack,cDarcy E. Hanes,b Mark A. Rasmussen,a* Maureen K. DavidsonaU.S. Food and Drug Administration, Center for Veterinary Medicine, Division of Animal and Food Microbiology, Laurel, Maryland, USAa; U.S. Food and Drug Administration,Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, Maryland, USAb; U.S. Food andDrug Administration, Center for Food Safety and Applied Nutrition, Division of Microbiology, College Park, Maryland, USAc; U.S. Food and Drug Administration, PacificRegional Laboratory Southwest, Irvine, California, USAd; University of California, Department of Microbiology and Molecular Genetics, Irvine, California, USAe; AgriculturalResearch Service, Bacterial Epidemiology and Antimicrobial Resistance Research Unit, Athens, Georgia, USAf; The Genome Institute, Washington University School ofMedicine, St. Louis, Missouri, USAg* Present address: Mark A. Rasmussen, Leopold Center for Sustainable Agriculture, Iowa State University, Ames, Iowa, USA.We report the draft genomes of Salmonella enterica subsp. enterica serovar Cubana strain CVM42234, isolated from chick feedin 2012, and S. Cubana strain 76814, isolated from swine in 2004. The genome sizes are 4,975,046 and 4,936,251 bp, respectively.Received 9 December 2013 Accepted 13 December 2013 Published 23 January 2014Citation Benahmed FH, Gopinath GR, Wang H, Jean-Gilles Beabrun J, Grim C, Cheng C-M, McClelland M, Ayers S, Abbott J, Desai P, Frye JG, Weinstock G, Hammack TS, HanesDE, Rasmussen MA, Davidson MK. 2014. Whole-genome sequencing of Salmonella enterica subsp. enterica serovar Cubana strains isolated from agricultural sources. GenomeAnnounc. 2(1):e01184-13. doi:10.1128/genomeA.01184-13.Copyright © 2014 Benahmed et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.Address correspondence to Maureen K. Davidson, Maureen.davidson@fda.hhs.gov.The presence of bacterial pathogens, such as Salmonella, in an-imal feed is an important route of animal infections and thus isan important risk factor for the human food chain and publichealth. There are 2,500 serovars of Salmonella, and all of themare considered a potential threat to human health (1). Salmonellaenterica subsp. enterica serovar Cubana has been reported in swinethat received contaminated feed (2) and in human foods (3).Whole-genome sequencing of Salmonella serotypes will allow thediscovery of new genes unique to Salmonella and will supportoutbreak investigations.The S. Cubana CVM42234 strain was isolated from chick feedwith the Bacteriological Analytical Manual Salmonella culturemethod (4, 5), detected by a quantitative PCR (qPCR) method (6,7), and serotyped with PCR serotyping (8) and by traditional se-rological methods according to the CDC protocols (9–11). Theserotype antigens are 1, 13, 23, and z29-. Pulsed-field gel electro-phoresis (PFGE) was performed according to the CDC methods(12), and the PFGE pattern was JDGX01.0018. In vitro antimicro-bial susceptibility testing was done according to the standard Na-tional Antimicrobial Resistance Monitoring System protocol us-ing the CMV2AGNF panel of antimicrobials (13). S. Cubanastrain 76814 was tested using a similar microbroth dilutionmethod (Trek Biosystems, Cleveland, OH). Strain CVM42234was susceptible to all antimicrobials tested, but strain 76814 wasresistant to streptomycin, sulfamethoxazole, and tetracycline.Strain CVM42234 DNA was extracted using the QIAcube(Qiagen, Valencia, CA), the DNA library was constructed accord-ing to the Illumina protocol with Nextera XT DNA Sample Prepkit, and it was sequenced using Illumina MiSeq (Illumina, SanDiego, CA). CLC bio software version 6.0.1 (Germantown, MD)was used for the trimming and de novo assembly of the paired-endreads to 100 contigs. Strain 76814 DNA was isolated with theGenElute isolation kit (Sigma-Aldrich, St. Louis, MO) for bacte-ria, the library was made by shearing and ligating Illumina se-quencing adapters to the genomic DNA, and it was sequenced onHiSeq 2000 (Illumina) then assembled using one Button Velvet(European Bioinformatics Institute, Hinxton, CambridgeshireUnited Kingdom) into 247 contigs (172 scaffolds).Both the CVM42234 and 76814 draft genomes were annotatedusing RAST (14). The two S. Cubana genomes have an averagenucleotide identity of 99.5% and reveal many common geneticfeatures, as well as some differences. They both have a multidrugresistancemdtABCD cluster, a multiple antibiotic resistance (mar)locus, and several multidrug resistance efflux pumps. Loci for re-sistance to metals, such as arsenic, copper, silver, cobalt, zinc, andmercury, are present. Many flagellar motility genes also are pres-ent. CVM42234 contains two discrete clusters of phage proteins. Aconjugative plasmid and a small plasmid bearing a mercury resis-tance locus were identified from the 76814 genome assembly. Thepresence of many antimicrobial resistance genes and strain-specific mobile elements indicate that S. Cubana may exhibit typ-ical heterogeneity based on host and geographical factors as seenin many other pathogenic Salmonella serovars. The annotations ofboth genomes are publically available at RAST.Nucleotide sequence accession numbers. The draft genomesequences for these two Salmonella serovar Cubana strains havebeen deposited at DDBJ/EMBL/GenBank under accession no.ATEU00000000 and AZGR00000000.ACKNOWLEDGMENTSWe thank Steffen Porwollik and Pui Cheng for their expert assistance.M.M. was supported in part by NIH grants no. AI039557, AI052237,Genome AnnouncementsJanuary/February 2014 Volume 2 Issue 1 e01184-13 genomea.asm.org 1AI073971, AI075093, AI077645, and AI083646, USDA grants no. 2009-03579 and 2011-67017-30127, the Binational Agricultural Research andDevelopment Fund, and a grant from the Center for Produce Safety.We thank Wen Lin for providing the Applied Biosystems 7500 Fastinstrument during this study, Venkatakrishna Shyamala for reviewing themanuscript, and Bill Klimki of the National Library of Medicine/NationalCenter for Biotechnology Information for assistance in submitting themetadata associated with this sequencing project.REFERENCES1. World Health Organization (WHO). 2005. Drug-resistant Salmonella.Fact sheet no. 139. World Health Organization, Geneva, Switzerland. http://www.who.int/mediacentre/factsheets/fs139/en/print.html.2. Osterberg J, Vågsholm I, Boqvist S, Lewerin SS. 2006. Feed-borneoutbreak of Salmonella Cubana in Swedish pig farms: risk factors andfactors affecting the restriction period in infected farms. Acta Vet. Scand.47:13–22. http://dx.doi.org/10.1186/1751-0147-47-13.3. Taormina PJ, Beuchat LR, Slutsker L. 1999. Infections associated witheating seed sprouts: an international concern. Emerg. Infect. Dis.5:626 – 634.4. Cheng CM, Lin W, Van KT, Phan L, Tran NN, Farmer D. 2008. Rapiddetection of Salmonella in foods using real-time PCR. J. Food Prot. 71:2436 –2441.5. Cheng C-M, Van KT, Lin W, Ruby RM. 2009. Inter-laboratory valida-tion of a real-time PCR 24-hour rapid method for detection of Salmonellain foods. J. Food Prot. 72:945–951.6. Andrews WH, Hammack T. 2007. Chapter 5, Salmonella. In Bacteriologicalanalytical manual online. U.S. Food and Drug Administration, Silver Spring,MD. http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm.7. AndrewsWH, Hammack TS. 2003. Chapter 1, Food sampling and prepara-tion of sample homogenate. In Bacteriological analytical manual online. U.S.Food and Drug Administration, Silver Spring, MD. http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063335.htm.8. Jean-Gilles Beaubrun J, Cheng CM, Chen KS, Ewing L, Wang H,Agpaoa MC, Huang MC, Dickey E, Du JM, Williams-Hill DM, Ham-ilton B, Micallef SA, Rosenberg Goldstein RE, George A, Joseph SW,Sapkota AR, Jacobson AP, Tall BD, Kothary MH, Dudley K, Hanes DE.2012. The evaluation of a PCR-based method for identification of Salmo-nella enterica serotypes from environmental samples and various foodmatrices. Food Microbiol. 31:199 –209.9. Grimont PAD, Weill FX. 2007. Antigenic formulae of the Salmonellaserovars, 9th ed. WHO Collaborating Centre for Reference and Researchon Salmonella. Institute Pasteur, Paris, France.10. Guibourdenche M, Roggentin P, Mikoleit M, Fields PI, Bockemühl J,Grimont PA, Weill FX. 2010. Supplement 2003–2007 (no. 47) to theWhite–Kauffmann–Le Minor scheme. Res. Microbiol. 161:26 –29. http://dx.doi.org/10.1016/j.resmic.2009.10.002.11. Centers for Disease Control and Prevention. 1988. Identification andserotyping of Salmonella. Frances W. Brenner Alma C. McWhorter-Murlin. Department of Health and Human Services, Centers for DiseaseControl and Prevention, National Center for Infectious Diseases, Divisionof Bacterial and Mycotic Diseases, Foodborne and Diarrheal DiseasesBranch, Foodborne and Diarrheal Diseases Laboratory Section, NationalSalmonella Reference Laboratory. CDC, Atlanta, GA.12. Centers for Disease Control and Prevention. 2002. Standardized molec-ular subtyping of foodborne bacterial pathogens by pulsed-field gel elec-trophoresis. Centers for Disease Control and Prevention, Atlanta, GA.13. U.S. Food and Drug Administration. 2011. National AntimicrobialResistance Monitoring System 2011 retail meat report. U.S. FDA,Silver Spring, MD. http://www.fda.gov/downloads/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/UCM334834.pdf.14. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA,Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, OlsonR, Osterman AL, Overbeek RA, McNell 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 usingSubsystems Technology. BMC Genomics 9:75. http://dx.doi.org/10.1186/1471-2164-9-75.Benahmed et al.Genome Announcements2 genomea.asm.org January/February 2014 Volume 2 Issue 1 e01184-13

Whole-genome sequencing of Salmonella enterica subsp. enterica serovar Cubana strains isolated from agricultural sources

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Whole-genome sequencing of Salmonella enterica subsp. enterica serovar Cubana strains isolated from agricultural sources

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