MobileGeneticElements2:2,72–80;March/April2012;G2012LandesBioscience The diversity of inducible and constitutively erm expressed (C) genes and association to different replicon types in staphylococci plasmids Lisbeth E. de Vries,1,2 Henrik Christensen1 and Yvonne Agersø2,* 1DepartmentofVeterinaryDiseaseBiology;UniversityofCopenhagen;Frederiksberg,Denmark;2NationalFoodInstitute;TechnicalUniversityofDenmark;Lyngby,Denmark Keywords: plasmid, phylogeny, erythromycin, tylosin, regulation . e t u The aim of this study was to analyze the diversity of the macrolide resistance gene, erm(C) in relation to structural b alterations affecting the gene expression. In addition, the association of erm(C) to mobile genetic elements (MGEs) in i r staphylococci mainly from Danish pigs was investigated. In total, 78 erythromycin-resistant isolates were screened for t s erm(C) by PCR. The erm(C) genes incl. the upstream regulatory region were sequenced and theexpression types were i characterizedphenotypically(agardiffusiontest)andgenotypically(sequenceanalysis).Phylogeneticanalysisoferm(C) d was compared with structural alterations affecting the gene expression. Plasmids carrying erm(C) from seven selected t isolateswerefullyorpartiallysequenced.Thirty-sevenisolateswereshowntobeerm(C)positiveanderm(C)frompigs o wereallconstitutively expressed,mainlycausedbydifferentsizeddeletions(118,111,107,70,66,16and3bp)inthe n regulatory region. Duplication (63 bp) and substitutions were also found to cause a constitutive phenotype. Only one o horseisolatehadaninducibleexpressiontype.Phylogeneticanalysisshowedthatstructuralalterationshavehappened D in different erm(C) allele groups and not only in one group. Furthermore erm(C) was found mainly on plasmids (~2.4–8kb)andgenesequencetypescorrelatedwithplasmidreplication(rep)genetypes.Oneerm(C)typewaslinkedto . anIS257elementabletocircularize.Inconclusion,structuralalterationsgivingrisetoconstitutiveexpressionoferm(C) e havehappenedseveraltimesintheevolutionoferm(C).Interestingly,thediversityoferm(C)appearstobelinkedtothe c n plasmidtype orMGEcarrying thegene. e i c s Introduction inducer,IR1pairswithIR2andIR3pairswithIR4inatwoloop o i structure which renders the erm(C) start codon non-accessible to B Staphylococciarepartofthenaturalskinfloraandcountforsome the ribosome, and only a leader peptide located just upstream of s of the most important veterinary pathogens, e.g., Staphylococcus erm(C) is translated.10 Upon induction, inducers bind to the e aureus and Staphylococcus hyicus.1 A very high prevalence of ribosome translating the leader, which leads to alteration in the d macrolide-lincosamide-streptograminB(MLS )resistanceisolates mRNAsecondarystructuresothatIR2pairswithIR3,theerm(C) B n hasbeenfoundamongstaphylococcal isolates fromanimals,2and start codon becomes accessible, and erm(C) is translated.10 a the erythromycin ribosome methylase (erm) gene erm(C) is the Structural alterations within the upstream region of erm(C) can L mostpredominantMLSBresistancegeneinstaphylococcalisolates result in constitutive expression of erm(C) which also confers 2 from both humans and animals.3,4 The erm(C) gene has mostly resistance to 16-membered macrolides (e.g., tylosin) and lincosa- been found on small multi-copy plasmids (2.3–2.5 kb),5 but also mides, streptogramin B and ketolides.7,9,14,15 Clinical reports and 1 0 larger and more diverse plasmids (3.7–4 kb) with mobilization in vitro studies have shown that the gene expression can change 2 (mob) and/or plasmid recombination (pre) genes have been from inducible to constitutive under selective pressure of non © reported.6-9 inducers.5,15-18 Although the use of non-inducing growth pro- The expression of erm(C) can be either inducible or con- moters such as tylosin was discontinued in Denmark after 1998, stitutive. Macrolide antibiotics are characterized by a lactone ring tylosin is still the predominant macrolide used for therapeutic containing 12–16 members, but only 14- and 15-membered treatment of infections in pigs.19,20 Alterations causing constitu- macrolides like erythromycin can induce erm(C) expression by tive expression of erm(C) are believed to be a result of high translational attenuation.10-13 This mechanism is controlled by concentrationsofnon-induciblemacrolideslikee.g.,tylosininthe theformationofahairpinstructureoftheerm(C)mRNAformed environment.5,16 bythepairingoffourinvertedrepeat(IR)sequenceslocatedinthe The literature contains a number of reports of structural region upstream of erm(C) (Fig.1A).10,12 In the absence of an alterations detected in the upstream regulatory region of *Correspondenceto:YvonneAgersø;Email:[email protected] Submitted:02/01/12;Revised:03/21/12;Accepted:03/22/12 http://dx.doi.org/10.4161/mge.20109 72 MobileGeneticElements Volume2Issue2 RESEARCHPAPER . e t u b i r t s i d t o n o D . e c n e i c s o i B s e d n a L 2 1 0 2 © www.landesbioscience.com MobileGeneticElements 73 Figure1(Seepreviouspage).Schematicrepresentationoftheinducibleerm(C)geneandstructuralalterationsintheupstreamregulatoryregion,which leadtoconstitutiveexpressionoferm(C).Theerm(C)mRNAbeginsat+1andbesidestheerm(C)gene(735bp)itcontainsanupstreamleaderORF(60 bp).Ribosomalbindingsites,ShineDalgarnosequence(SD)precedesbothgenes.Intheabsenceofaninducer,theinvertedrepeatsequences(IR),IR1- IR4formadoublehairpinstructure,renderingtheerm(C)startcodonnon-accessibletotheribosome.PredictedpairingofIRsequencesisindicatedwith arrows(thresholdforpredictedpairingwasDG,-41.84kJ/mol,-10kcal/mol)andhypotheticalpairingareindicatedwithdottedarrows.Structural alterationsthathavenotpreviouslybeenreportedarehighlightedwithanasterix(*).(A)Theupstreamregionoferm(C)fromtheS.aureusisolate (7504026–1)showntohaveaninduciblephenotypealignedwithtwoGenBanksequencesknowntohaveaninduciblephenotype.(B).Theregion upstreamerm(C)containingdeletionsresultinginaconstitutivephenotype.Thealignmentshowsthedifferentsizedeletionsfoundinsequencesfrom thisstudythathadaconstitutivephenotype.(C).Substitutions(underlinedinread)intheupstreamregionoferm(C)fromfourS.hyicusisolateswere predictedtoresultinthepairingofIR2withIR3,whichexplainstheconstitutivephenotype(seetext).(D)Theupstreamregionoferm(C)intheS.aureus isolate(65–5)predictedtohaveaconstitutiveexpressionoferm(C)duetoa63bpduplicationcontainingatruncatederm(C)´includinganadditional SD2´/IR4´. . e t erm(C).8,14-16Untilnow,threedifferenttypeshavebeenidentified Thirty-six S. hyicus and S. aureus isolates (35 from pig and 1 u (Fig.1): Sequence deletions of varying length, duplications of from horse isolates) had a constitutively expressed clindamycin b parts of the erm(C) gene as well as multiple point muta- resistance phenotype (Table 1; Fig. S1). Sequence analysis of the ri tions.8,14-16 However, no one has studied the evolution of the upstream regulatory region showed that 31 isolates contained st erm(C) gene in comparison with different structural alterations different size deletions (118 bp, 111 bp, 107 bp, 70 bp, 66 bp, i d causing a constitutive gene expression, and how the gene 16 bp and 3 bp), predicted to result in alternative secondary diversity is related to associated mobile genetic elements structures of the erm(C) mRNA, leaving IR4 and SD2 accessible ot (MGEs).Thus,theaimofthisstudywastoanalyzethediversity for translation of erm(C) (Fig.1B). Observed deletions of size, n of erm(C) in relation to structural alterations affecting the gene 118 bp, 111 bp and 107 bp included IR1-IR3. Deletions of size o expression,andtocharacterizeassociatedMGEsinstaphylococci 70 bp, 58 bp and 66 bp only included IR1, and pairing of IR2 D mainly from Danish pigs. Specifically, erythromycin resistant with IR3werepredictedtobemore stable(DG =-66.11kJ/mol, isolateswerescreenedfortheerm(C)gene,andaphenotypictest -15.80 kcal/mol) than the pairing of IR3 with IR4 (DG = . e revealed the erm(C) expression types. Sequence analysis -52.72 kJ/mol, -12.60 kcal/mol) (Fig.1A and B). Finally, c identified the genetic background for observed phenotypes. deletions of size 6 bp and 3 bp included all or part of IR3. No n Finally, phylogenetic analysis of the erm(C) gene were compared pairingofIR3containinga3bpdeletionwithIR4waspredicted, e with structural alterations causing a constitutive phenotype and since the calculated change in Gibbs free energy (DG -15.90 kJ/ i c with replicon types (rep gene) found on erm(C) carrying mol, -3.80 kcal/mol) was much higher than the threshold for s plasmids. pairing (, -41.84 kJ/mol, -10 kcal/mol) (Fig.1B). o However, four S. hyicus isolates (9805143-1, 9811071-1, i Results 7630009-4, 7430116-4) contained an upstream erm(C) region B with a complete leader sequence and four IR sequences, these s Screening S. aureus and S. hyicus isolates for erm(C). Out of were shown to have a constitutive phenotype. An agar disk e 78 erythromycin-resistant staphylococcal isolates, 16 S. aureus diffusiontestofanE.faecalisrecipient(JH2–2)transformedwith d and21S.hyicusisolateswereshowntobepositiveforerm(C)(all the plasmid carrying erm(C) from one of these four S. hyicus n 37 isolates are listed in Table 1). The highest prevalence of isolates (9811071-1), ruled out the possibility that another gene a erm(C) was found among S. aureus isolates from pigs, with could have caused the observed constitutive phenotype. The L 60.9% compared with 40.4% among S. hyicus isolates. sequence analysis showed that, for these four isolates, the 2 Expression types for erm(C). Agar diffusion tests showed that upstream erm(C) region contained four IR sequences with a few 1 only one S. aureus horse isolate (7504026-1) had an inducible substitutions (Fig.1C) that have not been characterized pre- 0 clindamycin resistance phenotype (Fig. S1), and sequence analy- viously, and these substitutions could explain the observed 2 sis showed the upstream regulatory region of erm(C) to be phenotype. The pairing of IR2:IR3 was predicted (DG = -83.68 © identical or highly similar to corresponding regions from isolates kJ/mol, -20.00 kcal/mol) instead of IR1:IR2 and IR3:IR4, thus known to have an inducible phenotype; S. aureus pT48 leaving the IR4 and SD2 accessible for translation of erm(C) (GenBank, M19652), pE5 (GenBank, M17990), pWG738 (Fig.1C).Finally,oneisolatethatwaslost(S.aureus65–5)could (GenBank, DQ088624),pE194 (GenBank, V01278),S.hominis not be tested phenotypically, but sequence analysis showed it to pSES5 (GenBank, Y09001) and S. lentus pSTE2 (GenBank, containa63bpduplicationincludinganadditionalSD2´/IR4´in AJ888003). Forthese sequences,IR1:IR2(DG= -44.43kJ/mol, the upstream regulatory region (Fig.1D). This erm(C) gene was -10.62 kcal/mol) and IR3:IR4 (DG = -52.72 kJ/mol, -12.60 predictedtobeconstitutivelyexpressedbythepairingofIR1:IR2 kcal/mol) were predicted to pair in the absence of an inducer (DG = -49.04 kJ/mol, -11.72 kcal/mol) and IR3:IR4´ (DG = with a total change in Gibbs free energy of DG = -97.15 -52.71 kJ/mol, -12.60 kcal/mol) with a total change in Gibbs total kJ/mol (-23.22 kcal/mol) (Fig.1A). In the presence of an free energy of DG = -101.75 kJ/mol (-24.32 kcal/mol) and total inducer, IR3 and IR4 were predicted to pair, DG = -66.11 leaving IR4 and the erm(C) start site accessible for translation kJ/mol (-15.80 kcal/mol) (Fig.1A). (Fig.1C). 74 MobileGeneticElements Volume2Issue2 Table1.Staphylococcistrainscarryingerm(C)usedinthisstudy Strain Species Source/year Otherresistance Approxsizeof erm(C) Expression GenBank phenotypes erm(C)plasmidor phylogenetic typeoferm(C)3 accession DNAring1 group2 number 9710013-1 S.hyicus pig/1997 STR 2.4kb 4a Constitutive JF968519 9730769-3 S.hyicus pig/1997 PEN,SPT,STR,SUL,TMP 1.7kb 2 Constitutive JF968541 9805143-1 S.hyicus pig/1998 PEN,STR,SUL,TET,TMP 3.7–4kb 1b Constitutive JF968536 9805598-14 S.hyicus pig/1998 - 2.4kb 4a Constitutive JF968536 9810320-1 S.hyicus pig/1998 PEN,STR,TET,TMP 1.7kb 2 Constitutive JF968538 9811071-1 S.hyicus pig/1998 PEN,SUL,TET,TMP 3.7–4kb 1b Constitutive JF968540 9831219-14 S.hyicus pig/1998 - 2.4kb 4a Constitutive JF968524 . e 9905227-14 S.hyicus pig/1999 PEN,STR 2.4kb 4a Constitutive JF968528 t u 9911527-1 S.hyicus pig/1999 - 2.4kb 4b Constitutive JF968522 b 9911757-14 S.hyicus pig/1999 PEN 2.4kb 4a Constitutive JF968529 i r 7213504-1 S.hyicus pig/2000 CIP,PEN,STR,TET,TMP 2.4kb 4a Constitutive JF968521 t s 7231274-2 S.hyicus pig/2000 - 2.4kb 4c Constitutive JF968517 i d 7313178-1 S.hyicus pig/2001 TET 2.4kb 4a Constitutive JF968543 t 7313624-1 S.hyicus pig/2001 PEN,STR,TET 2.4kb 4a Constitutive JF968520 o 7330561-6 S.hyicus pig/2001 PEN,SPT,STR,SUL,TET,TIA 2.4kb 4a Constitutive JF968514 n 7410443-1 S.hyicus pig/2002 PEN,SPT,STR 2.4kb 4c Constitutive JF968515 o 7411659-1 S.hyicus pig/2002 CIP,PEN,STR 2.4kb 4a Constitutive JF968516 D 7430116-4 S.hyicus pig/2002 PEN 3.7–4kb 1b Constitutive JF968534 . 7510871-1 S.hyicus pig/2003 PEN,STR 2.4kb 4a Constitutive JF968531 e c 7514773-1 S.hyicus pig/2003 PEN,TET 2.4kb 4a Constitutive JF968518 n 7630009-4 S.hyicus pig/2004 PEN,STR 3.7–4kb 1b Constitutive JF968535 e 7311242-1 S.aureus5CC398 pig/2001 SPT,STR,TET 2.4kb 4a Constitutive JF968512 ci 7312429-1 S.aureus pig/2001 SPT 2.4kb 4c Constitutive JF968510 s o 7411141-1 S.aureus5CC9 pig/2002 PEN,TET 4kb 3 Constitutive JF968526 i B 7413727-1 S.aureus5CC398 pig/2002 PEN,TET 2.4kb 4a Constitutive JF968513 7414035-2 S.aureus5CC398 pig/2002 PEN,STR,TET 2.4kb 4a Constitutive JF968509 s 7511314-2 S.aureus5CC398 pig/2003 SPT,TET,TMP 2.4kb 4a Constitutive JF968508 e d 7512166-1 S.aureus5CC398 pig/2003 PEN,STR,TET,TMP 7–8kb 1a Constitutive JF968537 n 7512986-1 S.aureus5CC398 pig/2003 SPT,TET 4kb 3 Constitutive JF968527 a 7504026-1 S.aureus5CC30 horse/2003 PEN,STR 2.4kb 4 Inducible JF968532 L 7612628-4 S.aureus5CC398 pig/2004 SPT,TET 7–8kb 1a Constitutive JF968539 2 200610584-1 S.aureus5CC9 pig/2006 PEN,TET,TMP 2.4kb 4a Constitutive JF968530 1 200640995-1 S.aureus horse/2006 PEN 2.4kb 4a Constitutive JF968511 0 2 65-5 S.aureus pig/2007 ND ND 4a Constitutive JF968533 © 66-1 S.aureus5CC9 pig/2007 - 2.4kb 4a Constitutive JF968507 71-1 S.aureus5CC398 pig/2007 PEN,TET,TMP 4kb 3 Constitutive JF968525 9b S.aureus5CC398 pig/2007 CEF,PEN,SPT,STR,TET,TMP 2.4kb 4a Constitutive JF968542 1Determined as the approximate size of the PCR product generated with primers pointing out of the erm(C) gene (primers 2020 and 2021 or 2022). 2Determinedbyaphylogeneticanalysis(Fig.2).3Determinedbyagardiffusiontest,exceptfor65-1thatwaspredictedtobeconstitutivelyexpressed.4PFGE patternof9805598-1wasshownbeidenticalwith9831219-1andthepatternof9905227-1wasshowntobeidenticalwith9911757–1(Fig.S5).5CCtype thatcouldbededucedfromspatypes(TableS1).IsolatespredictedtobeofCC398representeddifferentrelatedspatypessuchast034,t2876,t571.CEF, Ceftiofur; CIP, Ciprofloxaicin; CHL, chloramphenicol; ERY, erythromycin; FLO, florfenicol; PEN, penicillin; SPE, spectinomycin; STR, streptomycin; SUL, sulfamethoxazole;TET,tetracycline;TIA,tiamulin;TMP,trimethoprim;ND,notdetermined. www.landesbioscience.com MobileGeneticElements 75 . e t u b i r t s i d t o n o D . e c n e i c s o i B s e d Figure2.Phylogeneticgenetreeoferm(C)comparedwithstructuralalterationsintheupstreamregulatoryregionoferm(C)conferringconstitutive n expression.Sequencesfromthisstudy(markedinboldletters)weremainlyisolatedfrompigs(Table1),whereastheGenBanksequencesweremainly a fromhumanisolates.Structuralalterationscausingconstitutiveexpressionoferm(C)aremarkedwithacolorcode,andthetypeofalterationisnoted L withanumberwhichindicatesthesize(inbp)ofdeletionorduplicationcausingtheconstitutivephenotype.Bootstrapvaluesareindicatedatbranch 2 points(outof1,000generatedNJtrees).Therelativelylowbootstrapvaluesatsomebranchpointswithingroup4canbeexplainedbysinglenucleotide differencesbetweensequencesinthisgroup. 1 0 2 Phylogenetic analysis of erm(C) compared with expression The phylogenetic tree showed structural alterations in the © types. Comparing the erm(C) gene and the corresponding amino erm(C)upstreamregulatoryregiontobelocatedinallfourerm(C) acid sequences from all 37 staphylococcal isolates revealed 7 groups (Fig.2). Interestingly, inducible erm(C) genes were unique sequence types both on DNA and amino acid level. A only present within erm(C) group 3 and 4 (e.g., V01278; phylogenetic analysis of the 37 erm(C) gene sequences and 19 pE194andM19652;pT48/M117990;pE5)(Fig.2).Thuscom- erm(C)GenBanksequencesdividedthemintofourerm(C)groups plete regulatory sequences of inducible erm(C) genes have not (Fig.2).Acomparisonofthecorrespondingaminoacidsequences been reported for sequences belonging to erm(C) group 1 and 2. also resulted in the same grouping of four Erm(C) groups (data Overall, deletions appear to be the most common form of notshown).AsillustratedinFigure2,all56erm(C)genesequences alteration causing a constitutive erm(C) expression and specific were highly similar (95.1–100% DNA identity). The erm(C) type/sizes of deletions appear to be specific for the different sequenceswithingroups2,3and4werehighlyrelated(98.8–99% erm(C) groups. Only deletions of 16 bp were present in more DNA identity) whereas erm(C) of group 1 were more distinct thanoneoftheerm(C)groups;thehighlyrelatederm(C)group3 (95.1–95.9%DNAidentity)comparedwiththeothergroups. and 4 (Fig.2). Altogether, this showed that structural alterations 76 MobileGeneticElements Volume2Issue2 giving rise to constitutive expression have happened in different reflected in the prevalence of regulated erm(C) genes compared allele groups of erm(C) as opposed to in one type only. with constitutively expressed genes.16 In contrast, we found that Theerm(C)genesequencesarelinkedtotheplasmidcarrying allpigisolatescontainingerm(C)(1997–2007)wereconstitutively the gene. In order to test whether the sequenced erm(C) genes expressed, and therefore our results indicate that the therapeutic were located on small plasmids, which is most often the case,5-9 usageofnon-inducingmacrolideandlincosamidesinDenmarkstill PCR reactions using primers pointing in opposite directions out play an important role in selecting for constitutively expressed of erm(C) (Fig. S2), were performed. If erm(C) was located on a erm(C)genesinstaphylococcifrompigs.Alternatively,theseresults plasmid or a circular DNA molecule, PCR products would be maysimplyreflectthatthemainreservoiroferm(C)carryingstrains generated also when the primers were pointing in opposite were selected for constitutively expressed erm(C) genes before the directions.Inthiscase,differentsizePCRproductswereamplified banofgrowthpromoters,andthatthetherapeuticuseofmacrolides for 36 isolates (except S. aureus 65-5 that was lost during the and lincosamides continue to select for resistance genes such as study), and the product sizes were shown to correlate with the erm(C). It is worth noting that the only isolate found to have an . e fourerm(C)groups(Fig.2;Table1).Fullorpartialsequencingof inducible erm(C) expression was from a horse. This may be an t thesePCRproducts,representingeachofthefourgroups,showed indicationofthemuchlowerconsumptionofmacrolidesinhorses u erm(C) of group 1, 3 and 4 to be located on rep containing compared with pigs (in 2008, only about 1 kg of macrolides and b plasmids of approximately 2.4–8 kb, whereas erm(C) of group 2 lincosamidesweresoldfortherapeuticuseinhorsescomparedwith ri wasassociatedwithaIS257-liketransposasecontainingelementof the14,181kgsoldfortheuseinpigs).20Although,thiswouldhave st 1.7 kb (Fig. S3). tobeinvestigatedfurtherinastudydesignedtocompareexpression i d In order to study the relationship between erm(C) and asso- typesbetweendifferentanimalreservoirs. ciated plasmids, aphylogenetictreebasedonsequencedrepgenes Investigatingthegenetic basisof theobservedexpressiontypes, ot from this study and rep genes from erm(C) carrying plasmids we found mostly deletions of different size to be the cause of the n available from GenBank was constructed. The rep genes were constitutive phenotype. In this study, we identified structural o divided into three main groups of repU, repF and repL alterations causing constitutive expression of erm(C) that have D correspondingtoerm(C)phylogeneticgroup1,3and4respectively notpreviouslybeenreported.Theseincludeddeletionsof3bp,66 (Fig. 2; Fig. S4). Thus, the erm(C) gene sequences appear to be bp,118bp,a duplication of63bp,andnewsubstitutionswithin . e linkedtotheplasmid(orthemobileelement)carryingthem. theIRsequences.Thelatteralterationwasverifiedtoberesponsible c for the observed phenotype by transforming the S. hyicus plasmid n Discussion carryingerm(C)(9811071-1)intoanEnterococcusrecipientstrain. e This strongly suggests that the erm(C) located on the S. hyicus i c The screening of erythromycin resistant staphylococci showed plasmidwasresponsiblefortheobservedphenotype,eveniferm(C) s approximately half of the pig isolates to contain the erm(C) gene inductionmightworkslightlydifferentindifferentgenera. o (60.9% of S. aureus and 40.4% of S. hyicus) which is in overall The phylogenetic analysis illustrated that different types (sizes) i B agreement with former prevalence studies.2,3,21 of structural alterations causing a constitutive erm(C) expression Phenotypic testing revealed all pig isolates containing erm(C) may appear to be specific for the erm(C) groups, with the s to be constitutively expressed, and only one horse isolate was exception of 16 bp deletions that have occurred both in erm(C) e shown to contain an inducible erm(C) gene. In environments groups 3 and 4. However, a larger test population would be d with high concentrations of non-inducing macrolides (e.g., required to draw any broader conclusions from the observed n tylosin), staphylococci with constitutively expressed erm(C) genes trend. A former study showed the recombination system of a a are believed to have a selective advantage both to sensitive host cell to play a role in the development of different types of L staphylococci and to staphylococci containing a regulated erm(C) structural alterations associated with constitutive erm(C) gene 2 gene.5,16 Macrolides are one of the most commonly used anti- expression, and a model for the development of deletions in the 1 microbial agents fortherapeutictreatmentofinfectionsinpigsin erm(C) regulatory region by homologous recombination sug- 0 Denmark, and tylosin is the predominant macrolide used. Also gested the sequence region beginning just before IR3 until SD2 2 largeamountsofnon-inducinglincosamidesareusedinpigs.20In to be involved in a 16 bp deletion.14 The sequence in this region © 2008, a total of 14,181 kg macrolide and lincosamides were sold is identical in erm(C) group 2, 3 and 4 but differs from erm(C) for therapeutic use in pigs in Denmark. In comparison, only group1, which explainswhy 16bp deletionshave been observed about half of this use (7,600 kg of macrolide and lincosamides) inerm(C)group3and4,butnotingroup1.Replicationslippage wassoldfortherapeutictreatmentinpigsin1996(beforegrowth or illegitimate recombination may result in tandem duplications promoterswerediscontinuedinDenmark),buttheuseoftylosin observed in the erm(C) translational attenuator.15 Altogether, our for growth promotion alone was 68,350 kg. Evidence suggesting results showed that different types of alterations causing con- that the use of non-inducing macrolides (tylosin for growth stitutive expression of erm(C) have happened in different erm(C) promotion)andlincosamidesindomesticanimalsfromGermany, attenuators, and that the size and/or type of alterations that have UK and Denmark have selected for constitutively resistant happened appear to depend mainly on the sequence surround- erm(C)-carrying staphylococcal strains have also been reported.5 ing the alteration. However, it has also been suggested that the Another study has suggested that the discontinuation of tylosin frequency and type of alteration may depend on the selecting use for growth promotion after 1998 in Denmark could be antimicrobial agent.22 www.landesbioscience.com MobileGeneticElements 77 Interestingly, this study show that the erm(C) gene sequences the finding of erm(C) associated with an IS257 element on a are linked to the replicon type of the plasmid (or IS element) circular DNA element without any rep gene suggests a large carrying thegene.Wefounderm(C)tobelocated mainly (25/36 mobilization potential for this erm(C) gene, since it appears to isolates) on small repL containing plasmids of approx 2.4 kb, but havetheabilitytointegratebothinchromosomal,transposonand we also identified erm(C) on larger repF plasmids of approx. plasmids sites containing IS257 elements. 3.8 kb (3/36 isolates) and on repU plasmids of approx. 4–8 kb In summary, we have shown that erm(C) genes in Danish (6/36 isolates), also containing a pre/mob gene. In addition, staphylococcimainlyfrompigsareconstitutivelyexpressed,which erm(C)wasalsofound(for2/36isolates)tobeassociatedwithan ismainlycausedbydeletionsintheregulatoryregion.Alterations IS257transposase(alsoknownasIS431),anactivestaphylococcal giving rise to constitutive expression have happened in different insertion sequence23,24 previously found to be associated with allele groups of erm(C) and not only in one type, and the type other resistance genes in staphylococcus species,25 but this is the and/or size appear to be linked to the sequence surrounding the first report of erm(C) associated with IS257. Regarding the gene alteration. Furthermore, erm(C) was found to be located mainly . e regulations, it is worth noting that no complete regulatory onsmallplasmids,andthegenesequencewasshowntobelinked t sequence of an inducible erm(C) has been reported for erm(C) to the plasmid or the element carrying the gene. Altogether u group 1 and 2 linked to repU plasmids and IS257 transposase this suggests the different erm(C) carrying plasmids found in b elements, respectively. Thus it is not known whether possible staphylococci species until now have evolved independently. ri t structural alterations conferring constitutive expression for these s two erm(C) types may have occurred before or after the genes Materials and Methods i d were associated with their respective MGE. In fact, it remains to beprovenwhetherthesetwoerm(C)typeshaveactuallydeveloped Strains.The78erythromycinresistantisolatesfromDanishpigs, ot from an inducible gene type at all. horses and lamb used in thisstudy were identified as S.aureusor n As illustrated by the phylogenetic tree (Fig.2), erm(C) encod- S. hyicus as described previously.35 Different clones were repre- o ingplasmidshavebeendetectedindifferentstaphylococcalspecies sentedamongthestudiedisolatesasshownbyspatypingorPFGE D from various animal sources and also from humans. These typing(TableS1;Fig.S5).Allofthe52S.hyicusand20ofthe26 plasmids can be spread by mobilization between members of S. aureus were different diagnostic submissions to the National . different staphylococcal species, but also between staphylococci FoodInstitute,DTU(1997–2006),whichoriginatedfromfarms e c and bacillus.26-28 Most of the small erm(C) carrying plasmids, all across Denmark. Six S. aureus isolates were obtained from n however, do not encode any mobilization genes. In correlation differenthealthypigsfromatleasttwodifferentfarms(2007).As e with this, the phylogenetic erm(C) tree illustrates that within part of the standard procedure at DTU, all isolates were tested i c erm(C) group 4, three subgroups of identical erm(C) sequences for susceptibility to ceftiofur, chloramphenicol, erythromycin, s with identical size deletions in the upstream region have been florfenicol, penicillin, spectinomycin, streptomycin, sulfa- o detected both in S. aureus and S. hyicus (16 bp and 111 bp methoxazole, tetracycline and trimethoprim by using the broth i B deletion) and S. aureus, Bacillus subtilis and Neisseria meningitides microdilution Sensititer method (Trek Diagnostic Systems Ltd, (107 bp deletion). Thus, horizontal transfer of erm(C) on small UK) as described previously and following CLSI guidelines s plasmids without genes encoding mobilization has not only (Table1).36,37Duetoachangeinthestandardprocedure,strains e occurred between different species of staphylococci but also isolatedfrom 2000andlaterwerealsotestedforsusceptibility to d between bacteria from different genera. Such a transfer may have tiamulinandciprofloxacin(Table1).Twoisolates(9band71-1) n occurred by tranduction or transformation,29,30 or alternatively were positive for a mecA screening PCR, showing them to be a mobilization proteins encoded by other plasmids may facilitate methicillin-resistant S. aureus (MRSA) (http://www.crl-ar.eu/ L mobilization of small erm(C) plasmids carrying an oriT.31,32 The data/images/meca-pcr_protocol%2006.02.08.pdf) 2 disseminationisbelievedtobecontrolledbytheplasmidsstability PCR and sequencing. All the 78 erythromycin resistant 1 in the new hosts28 or alternatively by the occurrence of staphylococcal isolates were screened for erm(C) by PCR as 0 chromosome integration, which appears to be the case for erm described previously2 using DNA Taq polymerase (Ampliqon, 2 (C) observed in Neisseria meningitides. In this study we also Denmark) and the primers 28 and 29 (Table S2). The Bacillus © identified erm(C) on a mobilizable plasmid (repU) containing a subtilis strain B.3HU104 containing erm(C) on the plasmid pre/mob gene in S. hyicus. This plasmid was related to previously pE194 was used as a positive control for all PCR reactions. reported plasmids from Gram positive bacteria; with a pre/mob The erm(C) gene including an upstream regulatory region gene identical to pUB10, pC16, pG01, pSK41 and a recomb- (766-947 bp) of the 37 staphylococcal isolates that were positive ination site, RS (oriT) identical to a corresponding site in S. forerm(C)(Table1),wassequencedbyMacrogen, Korea (www. A saprophyticus plasmid pSES22 (AM159501). Interestingly, an macrogen.com/eng/sequencing/sequence_main.jsp)asoutlinedin almost identical region (1534 bp) from this S. hyicus plasmid, Figure S2. including erm(C) and starting just before the RS , (data not PCR reactions using the primers 2020 and 2021 or 2022 A shown) has also been detected on a large (20 kb) plasmid from pointing in opposite directions out of erm(C) (Fig. S2) were Lactobacillus reuteri (FJ489650) isolated from a pig in the performedfor36isolates(allisolatesthatwerepositiveforerm(C), 1970s.33,34 This strongly suggests that exchange of erm(C) except for S. aureus 65-5). PhusionTM High-Fidelity DNA between Lactobacillus and Staphylococcus has occurred. Finally, Polymerase (Finnzymes, Finland) was used with conditions 78 MobileGeneticElements Volume2Issue2 recommended by the manufacturer. Such PCR products were performed using QIAfilter Plasmid Midi Kit (Qiagen). completely or partially sequenced for seven isolates which were Electrocompetent Enterococcus faecalis, JH2-2RF cells were selected to represent different erm(C) phylogenetic groups as transformed with purified S. hyicus plasmid (9811071-1) or with determined by the phylogenetic analysis: For three S. hyicus anEnterococcuserythromycinresistantplasmid,PAT18(positive (7313178-1, 9730769-3, 9811071-1) and two S. aureus (9b, control), as described previously.45 Transformants were selected 7312429-1) isolates, the PCR products were completely onbrainheartinfusion(BHI)agarplates(Becton,Dickinsonand sequenced, and for two S. aureus (7512986-1, 7612628-4) the Company, USA), supplemented with 12.5 mg/L of rifampicin PCR products were partially sequenced. The following combina- and 12.5 mg/L of fusidic acid and either 4 mg/L or 8 mg/L tions of sequencing primers were used: For isolates 7512986-1 erythromycin. Altogether, 10 transformants (five from each the and 9730769-3, primers 2020, 2022 and ned234 were used, for BHI plates with 4 or 8 mg/L erythromycin, respectively) were isolates 7313178-1 and 9b, primers 2020, 2022, ned234 and selected and confirmed to carry a plasmid borne erm(C) by two gr4ned_b, for isolate 9811071-1 primers, 2020, 2021, op1SH, PCRs; one erm(C) PCR screen using primers 28 and 29, and a . e ned1, gr1upSH_b and gr1nedSH_SA_b and finally for isolate long PCR using the primers 2020 and 2021 pointing out of the t 7612628-4, primers 2020, 2021, gr1nedSH_SA_b and op1SA erm(C). For one transformant (JH2-2_9811071-1 B1-RFE), the u wereused.Allsequenceswereassembled,annotatedandvisualized PCR product from the long PCR (primer 2020–2021) was b in Vector NTI (Invitrogen). All primers used in this study are partiallysequencedwithprimer2021,andtheupstreamregionof ri t listed in Table S2. erm(C) was confirmed to be identical with the corresponding s Sequence analysis. GenBank was searched for full length region from donor S. hyicus (9811071-1) erm(C) plasmid. i d erm(C)genesbasedonthedefinitionthattheerm(C)gene shares Agardiffusiontestoferm(C)expressiontypes.For36staphy- $ 80% similarity on the amino acid level.4 Nineteen gene lococcalisolates(allisolateslistedinTable1,except65-5)andthe ot sequences were selected based on the following criteria; the 10 E. faecaelis transformants (inclusive JH2–2_9811071–1 B1- n upstream regulatory sequence should be sequenced and the RFE), expression types were tested by an agar disk diffusion test, o sequences should bepublished inapapercontaining information asdescribedpreviouslyandinaccordancewiththestandardCLSI D aboutthephenotypicerm(C)expressiontype.Aneighbor-joining disk diffusion method.46,47 For each of the tested strains, (NJ) tree based on a multiple alignment of the 37 erm(C) gene an erythromycin disc (15 mg) was placed in the middle . sequences (735 bp) obtained in this study and 19 erm(C) genes surrounded by two clindamycin discs (2 mg) (lincosamide e from GenBank was constructed in Clustal X38 and visualized by antibiotic) within a distance of 1–2 cm on a müller-hinton plate c n MEGA 4.0.2.39 The tree was rooted with the S. aureus erm(B) streaked with a standard 0.5 McFarland inoculum suspension. e gene (GenBank, AB300568) as outgroup. Another NJ tree based Plates were incubated for 16 to 18 h at 37°C. Flattening of the i on 5 replication (rep) genes that were identified on the full or clindamycin zoneadjacenttotheerythromycindisc (a“D-zone”) c s partial sequenced erm(C) plasmids from this study and 14 rep was considered proof for an inducible expression type. o geneslocatedonfullorpartialerm(C)containingplasmidsdeposit i in GenBank, was constructed as described above. Disclosure of Potential Conflicts ofInterest B Sequence analysis of the upstream region of erm(C) was No potential conflicts of interest were disclosed. s performed for the 37 isolates listed in Table 1 by alignment in e ClustalX38 and manual checking and editing of alignments in Acknowledgments d Bioedit version 7.0.0.40 Changes in Gibbs free energy (DG) for Part of this work was presented as a poster (B100) at the First n predicted pairing of IR sequences in the erm(C) mRNA were International ASM Meeting on Antimicrobial Resistance in a calculated with the program RNAfold (http://bibiserv.techfak. Zoonotic Bacteria and Foodborne Pathogens, 2008. We want L uni-bielefeld.de/rnafold/submission.html) using sequence regions to thank Christina Aaby Svendsen, Jacob Dyring Jensen and 2 containingthespecificIRsasqueries.41-43PairingofIRsequences Maria Louise Johannsen for excellent technical assistance. This 1 were predicted forDG ,-10 kcal/mol (-41.84 kJ/mol). All pair- work was funded by a grant from The Danish Research Council 0 wise sequence comparisons were performed with the EMBOSS for Technology and Production Sciences (274-05-0117). 2 program water (local alignments) and/or needle (global align- © ments) (http://www.ebi.ac.uk/emboss/align/).44 Supplemental Material Transforming a erm(C) plasmids into a recipient strain. Supplemental material may be found here: Plasmid purification from one S. hyicus isolate (9811071-1) was www.landesbioscience.com/journals/mge/article/20109 References 3. EadyEA,RossJI,TipperJL,WaltersCE,CoveJH, 4. Roberts MC, Sutcliffe J, Courvalin P, Jensen LB, Noble WC. Distribution of genes encoding erythro- Rood J, Seppala H. Nomenclature for macrolide 1. AarestrupFM,SchwarzS.AntimicrobialResistancein mycin ribosomal methylases and an erythromycin and macrolide-lincosamide-streptogramin B resistance StaphylococciandStreptococciofAnimalOrigin.In: efflux pump in epidemiologically distinct groups of determinants. 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