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This is the published version of a paper published in Journal of Global Antimicrobial Resistance.
Citation for the original published paper (version of record):
El Zowalaty, M E., Hickman, R A., Mthembu, T P., Zishiri, O T., El Zowalaty, A E. et al.
(2020)
Genome sequences of two Salmonella enterica strains (MEZSAL74 and MEZSAL81) harbouring multiple antimicrobial resistance genes isolated from livestock in South Africa
Journal of Global Antimicrobial Resistance, 21: 396-398 https://doi.org/10.1016/j.jgar.2020.04.001
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Genomenote
Genome sequences of two Salmonella enterica strains (MEZSAL74 and MEZSAL81) harbouring multiple antimicrobial resistance genes isolated from livestock in South Africa
MohamedE. ElZowalatya,*,RachelA. Hickmana,ThobekaP.Mthembub,OliverT. Zishirib, AhmedE.El Zowalatyc,JosefD.Järhultd
aZoonosisScienceCenter,DepartmentofMedicalBiochemistryandMicrobiology,UppsalaUniversity,Uppsala,Sweden
bDisciplineofGenetics,SchoolofLifeSciences,CollegeofAgriculture,EngineeringandScience,UniversityofKwaZulu-Natal,PrivateBagX54001,Durban 4000,SouthAfrica
cDepartmentofChemistry,UmeåUniversity,Umeå,Sweden
dZoonosisScienceCenter,DepartmentofMedicalSciences,UppsalaUniversity,Uppsala,Sweden
ARTICLE INFO
Articlehistory:
Received28January2020
Receivedinrevisedform2April2020 Accepted3April2020
Availableonline8May2020
Keywords:
Salmonella Africa Zoonosis
Antimicrobialresistance Multidrug
Genomesequencing
ABSTRACT
Objectives:Antimicrobial-resistantlivestock-associatedSalmonellaentericainfectionsposeasignificant public-health threat worldwide. Here we reportfor thefirsttimethedraftgenomesequencesoftwomultidrug- resistantlivestock-associatedS.entericastrainsisolatedfromachickenandacowinSouthAfrica.
Methods:Genomic DNA of S. enterica strains MEZSAL74 and MEZSAL81 was sequenced using an Illumina MiSeq platform.The generatedreads weretrimmedanddenovo assembled. Theassembled contigs were analysedfor antimicrobial resistance genes, chromosomal mutations and extrachromosomal plasmids. Multilocus sequencetyping(MLST)wasalsoperformed.InordertocompareisolatesMEZSAL74andMEZSAL81with otherpreviouslysequencedS.entericaisolates,rawreadsequencesweredownloadedandallsequencefiles weretreatedidenticallytogenerateabootstrappedmaximumlikelihoodphylogenetictree.
Results:Extrachromosomalplasmidsandgeneticdeterminantsofantimicrobialresistanceweredetected inbothsequencedbacterialisolatestoaminoglycosidesandfluoroquinolones.ByMLST,strainMEZSAL74 belongedtoanunknownsequencetype(ST)andstrainMEZSAL81belongedtoST33.
Conclusion:ThegenomesequencesofstrainsMEZSAL74andMEZSAL81reportedherewillserveasareference formolecularepidemiologicalstudiesofantimicrobial-resistantlivestock-associatedS.entericainAfrica.
©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobial Chemotherapy.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Salmonella enterica is a facultative intracellular foodborne pathogenofglobalimportancecausingmorbidityandmortalityin humansand that canbe transmittedthrough differentdistinct routes[1].Zoonoticsalmonellosisisagreatpublic-healthconcern.
Detectionof S.entericain livestock andfood-chainanimals has frequentlybeenreportedworldwideandrecentlyinSouthAfrica [1].Anincreasingnumberofstudieshavereportedtheacquisition of multiple antimicrobial resistances in S. enterica, including resistancetocolistin,posingaseriousthreattohumanandanimal health. The genome of S. enterica will provide important comparativegenomicinformation tohelpunderstanditspatho- genicityandtomonitoritsantimicrobialresistancecharacteristics.
ThereisscareinformationonthegenomesequenceofS.enterica isolatesfromAfrica.
S. enterica strains MEZSAL74 and MEZSAL81 were isolated from a cow and a chicken, respectively, in KwaZulu-Natal Province, South Africa, in May 2018. Agar slants of bacterial cultures were further analysed as part of the GenomeTrakr project. Isolateswere identified usingconventionalmicrobio- logicalmethodsforSalmonellaandwereconfirmedbycolonyPCR for the invA gene as previously described [1]. Samples were furtherprocessedforDNAisolationandwhole-genomesequenc- ing. DNA isolation was performed using a MasterPureTM DNA IsolationKit(LucigenCorp.,Middleton,WI,USA)accordingtothe manufacturer'sprotocol.Sequencinglibrarieswerepreparedusing aNexteraXTLibraryPrepKit(IlluminaInc.,SanDiego,CA,USA).
SequencingwasperformedonanIlluminaMiSeqplatformusinga v2ReagentKit (IlluminaInc.),whichyielded250-bp paired-end reads.
*Corresponding authorat:Zoonosis ScienceCenter,Department ofMedical BiochemistryandMicrobiology,UppsalaUniversity,Uppsala,SE75-123,Sweden.
E-mailaddress:elzow005@gmail.com(M.E. ElZowalaty).
http://dx.doi.org/10.1016/j.jgar.2020.04.001
2213-7165/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCCBY- NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
JournalofGlobalAntimicrobialResistance21(2020)396–398
ContentslistsavailableatScienceDirect
Journal of Global Antimicrobial Resistance
j o u r n a l h o m ep a g e: w w w . el s e v i e r . c o m / l o c at e / j g a r
Atotalof862.6Mband869.5Mbrawdatareadsweregenerated forMEZSAL74andMEZSAL81,respectively.Sequenceswereassem- bledusingUnicyclerv.0.4.7[2]into38contigsforMEZSAL74andinto 118contigsMEZSAL81.AssemblyqualitywasassessedusingQUAST [2]forbothisolates,yieldingthefollowing:forMEZSAL74,atotalof4 767316bpwithaGCcontentof52.12%,anN50of349646bpandan L50of4;andforMEZSAL81,atotalof4846596bpwithaGCcontent of52.19%,anN50of150971bpandanL50of10.Prokkav.1.13[3]was usedforannotationtoprovidevaluesoncodingsequences(CDS), tRNAs,tmRNAsandRNAs:fortheannotatedMEZSAL74genome therewere4465CDSand81tRNA,1tmRNAand3rRNAgenes;and fortheannotatedMEZSAL81genometherewere4554CDSand82 tRNA, 1tmRNAand3rRNAgenes.Toassesstheisolatesforgenomic epidemiologicalfactors,thecontigFASTAfileswereuploadedand analysedbythebacterialanalysispipeline-batchuploader[4].From theresults,onlyMEZSAL81hada knownsequencetype (ST33), thereforeweincludedforbothstrainstheclosestknownsequenced bacterialstrainanditsaccessionnumber;bothbacterialisolates alsohaveadetectedextrachromosomalplasmid(Table1).Antimi- crobialsusceptibilitytestingbythediskdiffusionmethodrevealed that MEZSAL74 was susceptible to amoxicillin/clavulanic acid (AMC),azithromycin,ceftriaxoneandchloramphenicol,interme- diate-resistanttociprofloxacinandresistanttosulfamethoxazole, tetracyclineandampicillin.MEZSAL81wassusceptibletoampicil- lin,AMC,azithromycin,ciprofloxacinandchloramphenicol,inter- mediate-resistanttoceftriaxoneandresistanttosulfamethoxazole andtetracycline.Formorein-depthcharacterisation,ResFinder3.2 [5]wasutilisedfordetectionofacquiredantimicrobialresistance genes and chromosomal mutations. Both strains contained acquiredantimicrobialresistancegenestoaminoglycosidesaswell asapreviouslyreportedchromosomalmutationinparC(T57S)that confers resistance to fluoroquinolones [6]; other chromosomal mutationswerealsodetectedthatcouldalsofurthercontributeto aminoglycosideandfluoroquinoloneresistancebuthavenotbeen previouslyreported(Table1).Thepresenceofthemutationsdidnot inducephenotypicresistancetofluoroquinolones;theonlyisolate toshowdecreasedsusceptibilitytociprofloxacinwasMEZSAL74,
which had intermediate resistance. No colistin or tetracycline resistance-conferringmutationsorcolistinortetracyclineplasmid- mediatedresistancemechanismswereidentifiedineitherstrain despitebothstainsexhibitingphenotypictetracyclineresistance.
This exemplifies that genetic determinants cannot always be demonstratedforallresistancephenotypes.
Inordertogenerateamaximumlikelihoodphylogenetictreeto establishhow thetwoisolates comparedwithotherpreviously sequenced S.enterica isolates,raw read fileswith origins from AfricafromBioProjectPRJNA293224(AppendixA;Supplementary Table S1) were downloaded. All raw read isolate files were processedbythesamemethodsoftrimminganddenovoassembly asmentionedabove.Allbacterialisolatefileswerethenannotated using Prokka v.1.13. Core genome alignments were done using Roary pangenomepipelinewithdefault setting. RAxMLv.8.2.12 was used for inference of bootstrapped maximum likelihood phylogenetictree,andvisualisationwasdoneusingthewebtool InteractiveTreeofLife(iTOL)[7](Fig.1).
StrainMEZSAL74wasphylogeneticallyrelatedtotheS.enterica subsp.entericaserovarBraenderupstrainSA20026289complete genome and was of unknown sequence type, whilst strain MEZSAL81 wasphylogenetically relatedtotheS.enterica strain FDAARGOS_313 complete genome and was ST33. The genome sequencepredictedtheserotypeofstrainMEZSAL81asserotype HadarandstrainMEZSAL74asserotypeAlachua.
Detection of antimicrobial resistance genes in livestock- associatedpathogensofzoonoticpotentialisaseriousincreasing public-healthconcern.Tothebestofourknowledge,thetwodraft genomesofstrainsMEZSAL74andMEZSAl81reportedinthisstudy arethefirstnon-human,livestock-associatedstrainsfromSouth Africa.Similarstudiesworldwidehavereportedthedetectionof antimicrobial resistance in livestock-associated S. enterica from diversesourcesofavian(chickenandturkey),swine,bovineand ovinehostsaswellastheirproductsandtheenvironment[8].This reporthighlights thesignificanceofcontinuedgenomicsurveil- lanceofnontyphoidalSalmonella,whichcontinuetoposeaserious global threat to human health and food security, primarily a
Table1
OverviewofSalmonellaentericastrainsMEZSAL74andMEZSAL81,detectedantimicrobialresistancegenes(ARGs)andassociatedchromosomalmutationswithpredicted phenotypes.
Strain Closestknown sequenced bacterialstrain accession number
Closestknown sequencedbacterial strain
Detected plasmid
Acquired ARGS
Identity (%)
Acquired ARG accession no.
Chromosomal mutation
Nucleotide change
Predicted resistance phenotype
Chromosomal mutationPMID reference
MEZSAL74 NZ_CP022490.1 S.entericasubsp.
entericaserovar Braenderupstrain SA20026289 chromosome, completegenome
IncY aac(60)- Iaa
97.26 NC_003197 Aminoglycosides
16SrrsD 1133A>G
A→G Aminoglycosides
16SrrsD 1139T>C
T→C Aminoglycosides
parCT57S ACC→AGC Fluoroquinolones 15388468 parCT255S ACC→TCC Fluoroquinolones
MEZSAL81 NZ_CP022069.2 S.entericastrain FDAARGOS_313 chromosome, completegenome
ColRNAI aph(3)-Ib 100 AF024602 Aminoglycosides
aph(6)-Id 100 M28829 Aminoglycosides
16SrrsD 92C>T
C→T Aminoglycosides
16SrrsD 249T>A
T→A Aminoglycosides
16S_rrsD 1133A>G
A→G Aminoglycosides
16S_rrsD 1139T>C
T→C Aminoglycosides
parCT57S ACC→AGC Fluoroquinolones 15388468 parCT255S ACC→TCC Fluoroquinolones
parCN395S AAC→AGC Fluoroquinolones parCS469A TCC→GCC Fluoroquinolones
M.E.ElZowalatyetal./JournalofGlobalAntimicrobialResistance21(2020)396–398 397
leadingcauseoffoodborneandinvasiveillnessesinhumans.This study highlights the importance of food-producing animals as principal reservoirs of many multidrug-resistant pathogenic S.
entericastrains.
Thiswholegenomesequencingprojecthasbeendepositedat GenBank/NCBIunderBioProjectno.PRJNA293224,withBioSample accessionnos.SAMN11636367andSAMN12058707andGenBank accessionnos.AAEAXB000000000and AAKADM000000000for strains MEZSAL74 and MEZSAL81, respectively. The versions describedinthispaperarethefirstversionsAAEAXB000000000.1 andAAKADM000000000.1.Thesequenceshavebeensubmittedto the Sequence Read Archive (SRA) under the accession nos.
SRR9050339andSRR9050348.
Ethicalapproval
This study was approved by the Animal Research Ethics Committee of the University of KwaZulu-Natal (Durban, South Africa)[referencenos.AREC/051/017M,AREC071/017andAREC014/
018;ActNo.35of1984Section20approvalreferenceno. 12/11/1/5].
Funding
ThisworkissupportedbytheUSNationalInstitutesofHealth/
Food and Drug Administration under award number 1U18FD006780-01 for whole-genome sequencing. In addition, theprojectwassupportedbytheSouthAfricanNationalResearch Foundation through Thuthuka Funding Instrument [grant no.
TTK170411226583] and the Swedish Research Council (Veten- skapsrådet)[grantno.2016-02606].
Competinginterests Nonedeclared.
Authors'contribution
M.E.Z. conceived, coordinated, and supervised the research project,preparedand wrotethemanuscript, andsubmitted the strainforWGS.M.P.andM.E.Z.conductedsamplescollectionand bacterialisolation.R.A.H.conductedthephylogeneticanalysis.R.A.
H.andJ.D.J.contributedtomanuscriptwriting.O.T.Z.,A.E.Z,andJ.D.
J. reviewed themanuscript. M.E.Z. criticallyrevised the manu- script.Allauthorsapprovedthefinalversionofthemanuscript.
Acknowledgements
WethankLyndyHardenandSiddharthaThakurfromPopulation Health and Pathobiology, College of Veterinary Medicine, North CarolinaStateUniversity(Raleigh,NorthCarolina,USA).Wethank theGenomeTrakrNetworkand theWhole GenomeSequencing ProgramforfoodbornepathogentracebackandtheCenterforFood Safety and Applied Nutrition (CFSAN), U.S. Food and Drug Administration (FDA), for their support in the whole-genome sequencing(WGS)ofthestrainsMEZSAL74andMEZSAL81aspart oftheUSFoodandDrugAdministration'sWGSsurveillanceeffort.
AppendixA.Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.jgar.2020.04.001.
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Fig. 1.Phylogenetic positioning of strains MEZSAL74 and MEZSAL81 (ST33) (highlighted inred) within Salmonellaenterica. Representative genomesof S.
enterica were usedto compare the two novel isolates. Maximum likelihood phylogenywasinferredfrom3346coregenesand16085totalgenes.Blackcircles representbootstrapbranchsupportvalues >90%based on100 replicates.ST, sequencetype.
398 M.E.ElZowalatyetal./JournalofGlobalAntimicrobialResistance21(2020)396–398
