Genomic characterization of multidrug-resistant extraintestinal pathogenic Escherichia coli isolated from grain culture soils

João Pedro Rueda FURLAN,Inara Fernanda Lage GALLO and Eliana Guedes STEHLING

Departamento de Análises Clínicas,Toxicológicas e Bromatológicas,Faculdade de Ciências Farmacêuticas de Ribeirão Preto,Universidade de São Paulo,Ribeirão Preto,São Paulo 14040-903(Brazil)

ABSTRACT Multidrug-resistant(MDR)Escherichia coli,mainly extraintestinal pathogenic E.coli(ExPEC),has been widely reported in infections worldwide.In agricultural soils,manure is a hotspot for the dissemination of antimicrobial resistance genes(ARGs)and pathogenic bacteria;however,MDR bacteria have also been reported in soils with no history of manure use.In addition,cross-resistance and co-resistance have been described as responsible for the metal-driven selection of bacteria resistant to antimicrobials.Therefore,the aim of this study was to analyze three MDR E.coli isolates obtained from Brazilian grain culture soil samples with no history of manure use by whole-genome sequencing.The MDR E.coli isolates were recovered from soils from corn and coffee fields,and presented resistance to β-lactams,quinolones,aminoglycosides,tetracyclines,sulphonamides,and dihydrofolate reductase inhibitor.Resistome analysis showed ARGs to several antimicrobials(i.e.,β-lactams,tetracyclines,aminoglycosides,sulphonamides,trimethoprim,phenicols,fosfomycin,and macrolides)as well as several metal resistance genes and antibacterial biocide resistance genes.In addition,known mutations in quinolone-resistance-determining regions of GyrA(Ser83Leu and Asp87Asn),ParE(Ser458Thr),and ParC(Ser80Ile)were also detected.Virulome analysis showed the presence of virulence genes(lpfA,mcmA,gad,mchF,iroN,cma,and iss)associated with ExPEC.Multidrug-resistant ExPEC isolates were assigned to phylogenetic group B1.The presence of MDR B1-ExPEC in soil samples shows the ability of these isolates to survive in soils.This study reports for the first time some sequence types(i.e.,ST345,ST448,and ST1146)of MDR E.coli in Brazilian soils.Therefore,these findings contribute to the monitoring of antimicrobial resistance and surveillance studies based on whole-genome sequencing worldwide.

Key Words: antimicrobial resistance gene,metal resistance gene,molecular typing,whole-genome sequencing

INTRODUCTION

In recent years,antimicrobial resistance has been classified as a global public health problem.This issue involves different spheres,including the environment,where studies are poorly performed compared with clinical settings(Aslamet al.,2018;Tiedjeet al.,2019).Antimicrobial resistance is a natural phenomenon,as antimicrobial resistance genes(ARGs)antedate the use of antimicrobials(D’Costaet al.,2011).Furthermore,different factors contribute to the spread of multidrug-resistant(MDR)pathogens to several sources.In the environment,the presence of clinically relevant ARGs such asβ-lactamase-encoding genes is worrisome and causing an increasing sense of public alarm(Heßet al.,2018),while in the clinical environment,MDR pathogens have already been reported to be associated with increased mortality rates in humans(Woodfordet al.,2011).

Multidrug-resistantEscherichia coli,mainly extraintestinal pathogenicE.coli(ExPEC),has been widely implicated in urinary tract infections of humans worldwide(Poolman and Wacker, 2016).Isolates ofE.coliare capable of surviving in soils and aquatic environments, and their MDR phenotypes have been reported in environmental samples from farms(Luoet al., 2011; Jones-Diaset al., 2016).In agricultural soils,the use of manure is a hotspot for ARGs and dissemination of pathogenic bacteria.However,MDR bacteria have also been reported in soils with no history of manure use(Williams-Nguyenet al.,2016;Morettoet al.,2020).

Some mechanisms,including cross-resistance and coresistance,have been described as responsible for the metaldriven selection of antimicrobial-resistant bacteria(Zhuet al.,2017).Co-existence of metal resistance genes(MRGs)and ARGs has been reported in non-contaminated and contaminated environments with antimicrobials and metals(Silveiraet al.,2014).It is well known thatE.coliis one of the most clinically relevant bacteria, which can accumulate ARGs,leading to an MDR profile.Thus, the aim of this study was to analyze three MDRE.coliisolates obtained from agricultural soil samples with no history of manure useviawhole-genome sequencing(WGS)and determine their resistome, virulome, sequence type (ST), and phylogeny.Therefore,this study can contribute to the monitoring and evaluation of the impact of MDRE.coliin agricultural soil samples.

MATERIALS AND METHODS

Bacterial isolation

Between 2018 and 2019,80 soil samples with no history of manure use were collected at a depth of up to 15 cm from several cities and different crops in Brazil.Firstly,bacteria were isolated from 1 g of each soil sample in 5 mL Brain Heart Infusion(BHI)broth(Oxoid Ltd.,UK).Subsequently,the cultures were streaked on MacConkey agar(Oxoid Ltd.,UK)(Furlanet al.,2019).A lactose-positive colony of each sample was stored in BHI and glycerol(15%)at−80°C for further experiments.

Identification of bacterial isolates

The PureLink™Genomic DNA Mini Kit(Thermo Fisher Scientific,USA)was used to extract genomic DNA.Molecular identification was carried out by 16S rDNA sequencing as described by Weisburget al.(1991).The MegaBLAST algorithm was used to analyze the obtained sequences(Morguliset al.,2008).

Antimicrobial susceptibility testing

The disk diffusion method was used to determine the antimicrobial resistance profile(CLSI,2019).A total of 33 antimicrobials were tested,including piperacillin-tazobactam,ampicillin-sulbactam,ampicillin,amoxicillin-clavulanate,imipenem,ertapenem,meropenem,cefazolin,ceftazidime,cefaclor, cefotaxime, cefuroxime, ceftriaxone, cefepime,aztreonam,streptomycin,gentamicin,amikacin,tobramycin,doxycycline,tetracycline,minocycline,trimethoprim,sulfonamides,trimethoprim-sulfamethoxazole,nalidixic acid,ofloxacin, levofloxacin, ciprofloxacin, norfloxacin, lomefloxacin,chloramphenicol,and nitrofurantoin(Cecon,Brazil).TheE.coliATCC®25922 andPseudomonas aeruginosaATCC®27853 were used as controls in this experiment,andE.coliisolates were classified as MDR following the criteria of Magiorakoset al.(2012),i.e.,resistant to one or more agents in three or more antimicrobial categories.

Metal tolerance profile

The metal tolerance profile was determined using 10-fold diluted trypticase soy agar(HiMedia, India)with selenite()(0.25,1.25,6.25,12.5,25,50,62.5,and 125 mmol L−1),nickel(Ni2+)(0.05,0.1,0.5,1,2.5,5,10,15,20,and 25 mmol L−1), silver (Ag2+) (5.8×10−3, 11.7×10−3,23.5×10−3,47×10−3,94×10−3,188×10−3,376×10−3, 752×10−3, and 1.5 mmol L−1), mercury (Hg2+)(10 and 50 mmol L−1), lead (Pb2+) (0.05, 0.1, 0.5, 1, 2,4,8,16,and 32 mmol L−1),cobalt(Co2+)(9.76×10−3,0.1, 1, and 5 mmol L−1)(Synth, São Paulo, Brazil), zinc(Zn2+)(5,10,20,and 50 mmol L−1),copper(Cu2+)(0.5,1,2,and 5 mmol L−1),chromium(Cr2+)(0.05,0.1,1,2,5,10,and 15 mmol L−1),cadmium(Cd2+)(0.6,1.25,and 2.5 mmol L−1),magnesium(Mg2+)(1,2,5,5,7.5,10,15,30,50,75,and 100 mmol L−1),iron(Fe3+)(0.1,0.5,1,2,4,8,16,32,64,and 128 mmol L−1)(Vetec,Brazil),tellurite()(0.25,1.25,6.25,12.5,25,50,62.5,and 125 mmol L−1)(Quimlab,Brazil),arsenic()(5,10,15,20,25,30,35,40,45,and 50 mmol L−1)(JT Baker,England),and manganese(Mn2+)(8×10−3,16×10−3,32×10−3,64×10−3,128×10−3,256×10−3,512×10−3,1,2,4,and 8 mmol L−1) (Hexis Científica, Brazil) (Deredjianet al.,2011).Klebsiella pneumoniaeEW158 was used as a control in this experiment(Furlanet al.,2020a).

Whole-genome sequencing and analysis

Whole-genome sequencing was performed on an Illumina MiSeq platform (250 bp paired-end) (Illumina Inc.,USA), andde novogenome assembly was carried out using CLC Genomics Workbench v.12(CLC Bio-Qiagen,USA).Virulence genes were analyzed using VirulenceFinder 2.0,ST and clonal complex(CC)using MLST 2.0,ARGs and mutations in quinolone resistance-determining regions(QRDR)using ResFinder 3.2,plasmids using PlasmidFinder 2.1,fimH-types using FimTyper 1.0, and serotypes using SerotypeFinder 2.0(http://www.genomicepidemiology.org/).Metal resistance genes and antibacterial biocide resistance genes (ABRGs) were analyzed using the BacMet database (http://bacmet.biomedicine.gu.se/).The identity statistics among MRGs and ABRGs were detected using sequence identity and similarity(Palet al., 2014).Phylogenetic groups were predicted using ClermonTyping(http://clermontyping.iame-research.center/).Prophage-related sequences were analyzed using PHASTER (Arndtet al.,2016).CRISPRs were detected using CRISPRCasFinder(Couvinet al.,2018).Genomic islands were identified using IslandViewer 4(Waacket al.,2006;Bertelliet al.,2017).The Blastmap was constructed using BRIG(Alikhanet al.,2011).Phylogenetic trees,core genome MLST,ribosomal MLST,and whole-genome MLST were analyzed using tools available at EnteroBase(Zhouet al.,2018,2020).

RESULTS

Escherichia coli isolates and WGS data

Among the 52E.coliisolates obtained from soils in São Paulo State, Brazil, 49 were susceptible to almost all tested antimicrobials and therefore were not classified asMDR.Three isolates(S526,S662,and S663)exhibited the MDR phenotype and were submitted to WGS.These isolates were obtained from soils of corn and coffee fields in three different cities(Altinópolis,Jardinópolis,and Sales Oliveira)(Table I).In those cities,a total of 20 samples were obtained,10 from Jardinópolis, 8 from Sales Oliveira, and 2 from Altinópolis.The mean GC content of the isolates sequenced by WGS was 50.6%,and the genome size ranged from 4.8 to 5.0 Mb.The number of genes, proteins, and GenBank accession numbers are listed in Table I.A Blastmap was constructed to compare the environmentalE.coliisolates andE.coliATCC®8739,a clinical isolate obtained from human feces(GenBank accession no.CP022959)(Blanco Massaniet al.,2018)(Fig.1).Some hypervariable genomic regions were detected,including ABC transporters,efflux systems,two-component regulatory system,DNA-binding proteins,mobile genetic elements,virulence factors,fimbrial complex, DNA-binding transcription regulators, type III secretion system,and genomic island(Fig.1).

Fig.1 Blastmap comparison of three E.coli isolates(S526,S662,and S663)and E.coli ATCC® 8739(GenBank accession no.CP022959),which was used as reference genome.The genomes and sequence similarity are demonstrated in different colors.

Antimicrobial resistance profile

All isolates were resistant to aminoglycosides (streptomycin), tetracyclines (tetracycline and doxycycline),βlactams (ampicillin, cefazolin, cefuroxime, and cefaclor),sulphonamides(trimethoprim-sulfamethoxazole and sulfonamides),dihydrofolate reductase inhibitor(trimethoprim),and quinolones(nalidixic acid,lomefloxacin,norfloxacin,ciprofloxacin, levofloxacin, and ofloxacin).Resistance to other aminoglycosides (gentamicin and tobramycin) was detected in two isolates(S662 and S663),while resistance to chloramphenicol and fosfomycin was detected only in the isolate S526.All isolates were classified as MDR(Table II).

Metal tolerance profile

The MDRE.coliisolates showed the same minimum inhibitory concentration (MIC) for selenite and tellurite(6.25 mmol L−1),cobalt and nickel(1 mmol L−1),arsenic(35 mmol L−1), chromium (2 mmol L−1), zinc (5 mmolL−1), copper (5 mmol L−1), mercury (>50 mmol L−1),cadmium(0.6mmol L−1),magnesium(>100 mmol L−1),lead (8 mmol L−1), manganese (4 mmol L−1), and iron(16mmol L−1).For silver, two isolates (S526and S662)presented an MIC of 23.5×10−3mmol L−1,while the third(S663)showed an MIC of 11.7×10−3mmol L−1.

TABLE IGenomic data for three multidrug-resistant E.coli isolates from grain culture soils in Brazil

TABLE IIPhenotypic and genotypic characteristics of three multidrug-resistant E.coli isolates(S526,S662,and S663)from grain culture soils in Brazil

Resistome and mobilome

Resistome analysis showed ARGs to tetracyclines(tetAandtetB),aminoglycosides(aadA1,aadA2,aph(3”)-Ib,andaph(6)-Id),β-lactams (blaTEM-1B), macrolides (mdf(A)),sulphonamides(sul1andsul2),phenicols(catA1),trimethoprim (dfrA5,dfrA8, anddfrA12), and fosfomycin (fosA);MRGs to arsenic(arsBCR),copper(pcoABCDERS,copAD,andnlpE),iron(fetAB),nickel(nikABCDER),silver(silABCEPRS),tellurium(tehABandterBC),magnesium/cobalt,(corAandmgtA), nickel/cobalt (rcnAB), tellurite/selenite(ruvB), and cadmium/zinc/cobalt (cusA); and ABRGs to quaternary ammonium compounds(QAC)(mdfA,sugE,andemrE)(Table II).The MRGs to copper(pcoABCDERS)and silver (silABCEPRS) were detected only in isolate S662,whereas the ABRG to QAC(emrE)was found only in theisolate S663.The ARGsblaTEM-1B,tetA,sul-like,dfrA-like,andmdf(A)were detected in all isolates.Other ARGs,such asaadA-like,were detected in isolates S526and S662,whileaph(6)-Idandaph(3”)-Ibwere found in isolates S662 and S663.The ARGscatA1andfosAwere detected only in the isolate S526.The resistance phenotype profile corroborates with the genotypes,includingβ-lactams,aminoglycosides,tetracyclines,sulphonamides,trimethoprim,and phenicols(Table II).

Mutations in the QRDR of GyrA (Ser83Leu and Asp87Asn),ParE(Ser458 Thr),and ParC(Ser80Ile)were also detected.Mobilome analysis revealed the presence of different plasmids,including IncF(pMLST 2,29,and 24),IncFIB1, pXuzhou21, and Col (MG828) (Table II).The nucleotide sequences of MRGs and ABRGs shared in all MDRE.coliisolates were analyzed for identity,except for

pcoABCDERS,silABCEPRS,andemrE,which were identified only in isolates S662 and S663,respectively.The mean percentage of identity ranged from 96.84%to 100%.Two genes(fetAandfetB)showed 100%identity.Some MRGs and ABRGs(arsB,arsR,copD,nlpE,terB,terC,nikB,nikD,

nikE,rcnB, andsugE) showed 100% maximum identity(Table III).Isolates S526and S662 shared the largest number of identical MRGs and ABRGs(arsB,nlpE,nikE,rcnB,andsugE)followed by S526and S663(copD,terB,nikB,andnikD),and S662 and S663(arsRandterC)(Table III).

Virulome

Virulome analysis showed the presence of genes encoding long polar fimbriae(lpfA),microcin M part of colicin H(mcmA),glutamate decarboxylase(gad),ABC transporter protein MchF(mchF), enterobactin siderophore receptor protein(iroN),colicin M(cma),and increased serum survival protein(iss)(Table II).Two virulence genes(i.e.,gadandlpfA)were found in all isolates.Multidrug-resistantE.coliisolates harbored 5–9 CRISPR and 38–50 genomic islands(Table II).A prophage-related sequence was detected in each MDRE.coliisolate.

Escherichia coli typing and epidemiological analysis

All MDRE.coliisolates were assigned to phylogenetic group B1.The MLST analysis revealed different STs,CCs, andfimH-types, such as ST1146-fimH369 (S526),ST345/CC23-fimH31 (S662), and ST448/CC448-fimH35(S663).Furthermore,three different serotypes were detected,including O185:H2(S526),O134:H53(S662),and O8:H8(S663)(Table II).Isolate S526(B1-ST1146-fimH369)was not grouped with the fewE.coliisolates from Sweden,UK,and USA(Fig.S1,see Supplementary Material for Fig.S1).Isolate S662(B1-ST345-fimH31)was grouped together with human,farm animals,food,and plantE.coliisolates from USA,Spain,Australia,Nigeria,Germany,and China(Fig.S1),whereas isolate S663(B1-ST448-fimH35)was grouped in a cluster only with clinicalE.coliisolates from USA and The Netherlands(Fig.S1).

DISCUSSION

This study reports the presence of MDR B1-ExPEC carrying ARGs and MRGs in grain culture soils that are widely used for animal and human feeding,and consequently can spread to other sources.Among the phylogenetic groups of ExPEC,B2 is the most prevalent,while other phylogenetic groups(i.e.,A and B1)are reported as commensal isolates.However,B1-ExPEC has been increasingly reported to cause infections worldwide, including Brazil (Clermontet al.,2013; Chakrabortyet al., 2015; Cyoiaet al., 2015).The MDRE.coliisolates have been detected for many years,mainly among clinical isolates,and MDR ExPEC has alreadybeen reported in humans,livestock,companion animals,and wildlife(Ewerset al.,2012).Recently,more attention has been given to the detection of MDR pathogens as well as ARGs in environmental samples(Furlan and Stehling,2017).

TABLE IIIIdentity statistics(IS)among metal resistance genes and antibacterial biocide resistance genes found in multidrug-resistant E.coli isolates from soils in Brazil

In Brazil,some STs of MDRE.coliisolates(A-ST189-fimH54 and B1-ST906-fimH61)were recently reported for the first time in soils cultivated with jaboticaba and guava(Furlan and Stehling,2019a,b).These isolates and others isolated from water sources possessedgadandlpfAgenes,which are involved in bacterial survival in acidic environments and adherence,and may be associated with survival in environmental spheres(Furlanet al.,2020b).The MDRE.coliS526was assigned to ST1146,a rare ST mainly detected in animals used for human consumption(Bortolaiaet al.,2011; Zurfluhet al., 2014).TheE.coliST1146isolates harboringblaCTX-M-1were reported in poultry(Zurfluhet al.,2014),while in the environment,anE.coliST1146isolate was reported in freshwater from a shellfish-harvesting area in France(Balièreet al.,2015).Fluoroquinolone-resistantE.coliST1146carryingblaCTX-M-55was detected in healthy humans in Thailand(Seenamaet al.,2019).Intriguingly,E.coliST1146isolates have also been isolated from fruit bats in Africa(Nowaket al.,2017).

The MDRE.coliS662 belongs to ST345,an international clone that has been reported worldwide carrying several ARGs,mainly extended-spectrumβ-lactamase encoding genes (Casellaet al., 2017; Boehmeret al., 2018; Touatiet al., 2020).TheE.coliST345 harboringblaCTX-M-3andblaTEM-1Bwas detected in a dog from Germany(Boehmeret al.,2018).In South America,including Brazil,E.coliST345 isolates carryingblaCTX-M-likewere previously found in commercial chicken meat and wild owls(Casellaet al.,2017;Fuentes-Castilloet al.,2019).Recently,mcr-1-producingE.coliST345 was reported in irrigation water from the North West Algerian farmlands (Touatiet al., 2020).Sequence types ofE.colibelonging to CC23,including ST345,isolated from humans and animals,are frequently associated with multidrug resistance, including resistance to fluoroquinolones andβ-lactams,which corroborates the profile of MDRE.coliS662(Giufrèet al.,2012;Haenniet al.,2014).

The MDRE.coliS663 was identified as belonging to ST448,a widespread human-associated clone that has been reported to cause different types of infection(Pitartet al.,2015;Muggeoet al.,2020).New Delhi metallo-β-lactamaseproducingE.coliisolates belonging to ST448 were recovered from clinical specimens in Cuba and Kuwait(Aunget al.,2018;Quiñoneset al.,2020).Fluoroquinolone-resistantE.coliST448 isolates have been reported in humans and food products from Nigeria and Germany(Chattawayet al.,2016;Irrganget al., 2017).Recently, anE.coliST448 isolate carryingblaCMY-2was reported for the first time in Brazil from a human infection(Kogaet al.,2019).To the best of our knowledge,there are no reports on soil samples of the STs detected in this study.

Bacteria harboring MRGs are emerging as a global health problem because metals in soils may contribute to the selection of MDR bacteria,includingE.coli(Deuset al., 2017).Similar phenotypes and genotypes for metals,including MICs and MRGs for copper,arsenic,zinc,cobalt,magnesium,cadmium,selenium,tellurium,and nickel,were found inK.pneumoniaeisolates recovered from aquatic environments(Furlanet al.,2020a).The different percentages of identity among the MRGs and ABRGs detected in all MDRE.coliisolates may be related to the frequent genetic recombination and exposure to compounds such as metals(Wales and Davies,2015).The genomic islands have been related to microbial adaptability and genome evolution(Juhaset al.,2009).Furthermore,some genetic elements,such as multidrug resistance plasmids,have been closely associated with the rapid spread and development of MDRE.coli(Carattoli,2013).IncFplasmids,which are mainly associated with ARGs,were detected in all isolates in this study.They have been described as the most common plasmid family found inE.coliisolates(Yanget al.,2015).

CONCLUSIONS

The presence of MDR B1-ExPEC in soil samples shows the ability of the isolates analyzed in this study to survive in soils.The detection of international human-associated clones calls attention to the dissemination of these isolates in the environment.This study reports for the first time three STs(ST345,ST448,and ST1146)of MDRE.coliin Brazilian soils.Therefore,these findings contribute to the monitoring of antimicrobial resistance and surveillance studies based on WGS worldwide.

ACKNOWLEDGEMENT

This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Nos.2018/19539-0 and 2018/01890-3)and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Nos.88882.180855/2018-01 and Finance code 001),Brazil.

SUPPLEMENTARY MATERIAL

Supplementary material for this article can be found in the online version.