The! worldwide! increase! in! penicillin! resistance! has! coincided! with! an! increase! in! macrolide!resistant!pneumococci.!In!many!parts!of!the!world!macrolide!resistance!rates! now! exceed! that! of! penicillin! resistance.! PROTEKT! data! (1993H2003)! shows! a! global!
! PatA! PatB! ! ! ATP FQ ADP$+$Pi Cell$membrane!
OUT!
IN!
increase! in! macrolide! resistance,! from! 31%! in! 1999! to! 36.3%! in! 2003.! Resistance! rates! fluctuate! greatly! between! countries,! with! rates! a! low! as! 3.9%! reported! in! the! Czech! Republic!while!rates!as!high!as!91.2%!have!been!reported!in!Taiwan!(126).!A!recent!trend! is!the!escalation!of!macrolide!resistance!in!countries!typically!associated!with!low!rates!of! resistance!due!to!the!dissemination!of!antimicrobial!resistance!clones;!in!South!Africa!the! incidence!of!macrolide!resistance!increased!from!9%!in!2000!to!14%!in!2005!due!mainly! to! the! dissemination! of! MDR! clones! (42).! In! Malawi! in! contrast,! resistance! to! the! macrolide,!erythromycin!has!consistently!remained!below!2%!over!the!last!decade!(78).! !
Macrolides!are!microbiostatic!agents!that!inhibit!bacterial!protein!synthesis!by!binding!to! the! 23S! ribosomal! RNA! and! include! azithromycin,! clarithromycin,! erythromycin! and! telithromycin.! The! two! main! mechanisms! of! macrolide! resistance! in!S.* pneumoniae! are! modification! of! the! target! site! and! efflux! of! the! antimicrobial! from! the! cell,! through! acquisition!of!the!erm!or!mef!genes!respectively!(127).!
1.7.6 erm!mediated!resistance!
Acquisition! of! the!erm(B)! gene! encoding! a! 23S! RNA! methylase! is! a! major! resistant! determinant! in! pneumococci.! Expression! of!erm(B)! results! in! the! posttranscriptional! modification! of! 23S,! reducing! the! affinity! of! the! macrolide! to! the! 23S! binding! site.! The! majority!of!pneumococci!that!encode!erm(B)!exhibit!the!‘MLSB!phenotype’;!resistance!to!
14H,!15H!and!16H!member!ring!macrolides,!lincosamides!!and!streptogramin!B.!High!level! macrolide!resistance!(>64µg/ml)!is!generally!associated!with!mutations!in!erm(B)!(128).!! More!recently!a!novel!erm!gene,!erm(TR)!has!also!been!shown!to!confer!MLSB!resistance!
(129)!.! !
In! pneumococci,!erm(B)! is! carried! on! members! of! the! Tn916! family! of! transposons.! A! number! of! Tn916! derivatives! carrying! erm(B)! have! been! described! in! pneumococci! (Tn3872,! Tn6002,! Tn6003,! and! Tn1545)! and! these! transposons! also! encode! the!tet(M)! gene! conferring! tetracycline! resistance.! As! a! result! there! is! a! high! incidence! of! tetracycline!resistance!amongst!macrolide!resistant!pneumococci.!It!should!however!be! noted!that!the!tet(M)!gene!is!not!always!expressed!(130).!
One! of! the! earliest! derivatives! of! Tn916! described! was! Tn3872,! a! 23.3k! composite! element!resulting!from!the!insertion!of!the!erm(B)!carrying!element,!Tn917!into!Tn916* (131).!The!20.9kb!Tn6002!is!the!result!of!the!insertion!of!a!2.8kb!DNA!fragment!encoding! erm(B)!into!Tn916*(132).!Tn6003,!a!25.1kb!transposon,!is!the!result!of!the!insertion!of!the! 4.4kb! macrolideHaminoglycosideHstreptothricin! (MAS)! element! into! Tn6002.! The! MAS! element!contains!aphAH3!and!an!additional!erm(B)!and!as!a!result!Tn6003!encodes!two! erm(B)! genes! (130).! The! first! derivative! of! Tn916* described! was! Tn1545,! a! 25.3kb! transposon!detected!in*S.*pneumoniae!BM4200!(133).!Recent!characterisation!has!shown! that!Tn1545!is!actually!1kb!larger!than!originally!reported,!the!transposon!results!from! the!insertion!of!the!1200bp!putative!insertion!sequence!IS1239!into!Tn6003.*In!addition! to! macrolide! and! tetracycline! resistance! Tn1545! and! Tn6003! confer! resistance! to! kanamycin!and!structurally!related!aminoglycosides!through!the!presence!of!the!aphAH3! gene!on!the!MAS!element!(134).!!
!
The!macrolide!resistance!determinant!erm(TR),!a!subclass!of!erm(A)!was!first!identified!in! S.* pyogenes*(129).! In! 2001! the! first! documented!erm(TR)! carrying! pneumococcal! strain! was! isolated! from! two! Greek! patients! (135).! In! contrast! to!erm(B),!erm(TR)! carriage! appears!to!be!rare!in!pneumococci.!Analysis!of!over!1000!pneumococcal!strains!from!25! countries!found!erm(TR)!was!present!in!only!2!strains,!both!from!Australia!(136).!Given! the!high!frequency!at!which!erm(TR)!is!present!in!S.*pyogenes,!erm(TR)!could!be!expected! to!circulate!more!readily!to!pneumococci!in!the!nasopharynx!(137).!Characterisation!of! the! erythromycin! resistant,!S.* pneumoniae! AP200! revealed!erm(TR)! was! carried! on! a! relatively!large!~56.0kb!genetic!element!designated,!Tn1806.!It!is!possible!that!the!size!of! Tn1806!means!that!it!is!difficult!to!acquire!and!maintain!in!the!species!(138).!!
!
There!is!extensive!evidence!documenting!the!dissemination!of!ermHmediated!resistance! by!clonal!spread.!Analysis!of!109!Spanish!erythromycin!resistant!pneumococci!exhibiting! the! MLSB! phenotype! showed! that! five! clones! (Sweden15AH25,! clone19FHST276,! Spain23FH1,!
Spain23FH2! and! clone19AHST276)! account! for! over! 50%! of! all! MLSB!strains.! Furthermore,!
77.1%! of! MLSB! strains! possess! genes! relating! to! Tn916,! suggesting! that! macrolide!
resistance!is!being!disseminated!through!the!clonal!spread!of!Tn916!family!transposons! (139).!
Figure!1.8!Representation!of!Tn916!and!related!elements!in!S.*pneumoniae!
!!
The! arrowed! boxes! represent! coding! sequences! (CDSs)! and! their! direction! of! transcription.!The!colours!of!the!CDSs!indicates!the!predicted!function!of!the!protein!it! encodes:!blue,!conjugation;!green,!transcriptional!regulation;!grey,!insertion!and!excision;! red,! accessory! genes.! ! Genes! absent! from! Tn916! are! shown! in! orange! in! the! other! elements.!Taken!from:!Roberts!&!Mullany!2011,!(140)!
!
1.7.7 mef!mediated!resistance!
Acquisition! of!mef! genes,! encoding! an! active! (proton! dependent)! efflux! pump! is! the! second! major! mechanism! of! macrolide! resistance.! The! majority! of! mef! positive! pneumococci! exhibit! the! ‘M! phenotype,’! characterised! by! resistance! to! 14H! and! 15H! member! ring! macrolides.! The! M! phenotypes! exhibit! lower! erythromycin! MICs! than! the! MLSB!phenotype,! 1H32µg/ml! in! comparison! to! >32µg/ml! (74).! There! are! at! least! three!
subclasses!of!mef;!the!abundant!mef(A)!and!mef(E),!which!share!90%!sequence!identity! and!the!rare!variant!mef(I)!which!has!only!been!described!in!two!Italian!clinical!strains! (141).!The!incidence!of!mef(A)!and!mef(E)!varies!greatly!from!country!to!country,!mef(A)! dominates!in!Europe,!while!mef(E)!dominates!in!the!United!States,!Asia!and!South!Africa! (142)!.!
!
In! pneumococci! the! three! subclasses! of!mef! are! carried! on! a! number! of! similar! but! distinct!genetic!elements.!All!of!these!elements!also!encode!a!msr(D)!gene!that!encodes! been reported inS. aureus. Transfer of plasmid-located and
chromosomal copies of Tn916fromE. faecalisISP1047 toS. aureus has been demonstrated (Joneset al., 1987). Addi- tionally a survey of 37 tet(M) containing staphylococcal strains demonstrated, by dot blot hybridization, that two containedintTn(Poyart-Salmeronet al., 1991). Sequencing of the methicillin-resistant strain Mu50 revealed the pre- sence of a putative conjugative transposon that was related to Tn916 and designated Tn5801 (Kuroda et al., 2001) (Fig. 1d). A comprehensive survey of 205 tetracycline- resistantS. aureusstrains isolated from humans and animals (cattle, lamb and pigs) were screened for the presence of
tet(M) and the integrase of either Tn916or Tn5801(which share 38.6% identity at the nucleotide level). Tn916-like elements were found in isolates from both humans and animals, whereas Tn5801was found only in isolates from humans (de Vries et al., 2009). Additionally, one of these Tn5801-like elements was shown to transfer and designated Tn6014(de Vrieset al., 2009). There are multiple alleles of
tet(M), each of which is usually associated with one type of mobile element; for example there is a tet(M) allele asso- ciated with Tn916and anothertet(M) allele associated with Tn5801(de Vrieset al., 2009).
Tn916-like elements in S. pneumoniae Tn1545 was initially discovered in S. pneumoniae strain BM4200. The element is essentially Tn916with an insertion of the erm(B) gene encoding macrolide, lincosamide and streptogramin (MLS) resistance and the kanamycin resis-
tance gene aphA-3 (Courvalin & Carlier, 1986; Cochetti
et al., 2008). Many clinical strains ofS. pneumoniaecarry
tet(M), which is usually resident on Tn916/Tn1545-like elements; eight out of the 36 pneumococcal genomes currently sequenced contain one of these elements (Santoro
et al., 2010). Since then various Tn916/Tn1545-like elements have been detected and characterized inS. pneumoniae or found in other streptococci. Early work aimed at detecting Tn1545-like elements by identification of the various resis- tance genes present in isolates by dot blot (Seralet al., 2001) showed a range of combinations of the genes present on Tn1545. Additionally, 63 out of 65 S. pneumoniae strains showed the presence ofintTndemonstrating, that elements from the Tn916/Tn1545group were likely to be common in this organism (Montanariet al., 2003). It is likely that this early work was in fact detecting some of the more recently characterized conjugative transposons such as Tn6002and Tn6003. Tn6002was initially characterized inStreptococcus cristatus from a clinical sample taken from a periodontal patient (Warburtonet al., 2007). The element is essentially Tn916 with an insertion in orf20 (Fig. 3). The insertion contains five genes, one of which iserm(B) conferring the MLS phenotype upon its host (Warburton et al., 2007). Subsequently, Tn6002-like elements were found in many strains ofS. pnuemoniaecollected in Italy between 2000 and 2002 (Cochetti et al., 2007). These authors also found a derivative of Tn6002, which contained an additional insert of the macrolide–aminoglycoside–streptothricin (MAS) ele- ment within theerm(B) gene cluster of Tn6002(Cochetti
et al., 2007) (Fig. 3). Further analysis of the BM4002
Tn916 ermB Tn3872, Tn2017 Tn2009, Tn2010, Tn2017 Tn1545 Tn6002,Tn6003,Tn1545,Tn2010 Tn6003,Tn1545 ermB aphA-3 ermB mef (E) IS1239
Fig. 3.Schematic representation of the diversity of mobile elements associated with Tn916inStreptococcus pneumoniae. The colours are as in Fig. 1. The additional resistance genes are labelled vertically. The elements in which each insertion is found is labelled near the insertion; note that some insertions are present in more than one element and some elements, for example Tn2010, contain more than one insertion.
FEMS Microbiol Rev35(2011) 856–871 !c2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 865 Diversity of Gram-positive Tn916elements
insertion event has led to the possible production of a fusion protein (Sebaihiaet al., 2006). Outward reading promoters from integrated elements can lead to differential expression of host genes. This has been observed experimentally for
Tn916 in B. subtilis (Celli & Trieu-Cuot, 1998). Further-
more, in E. faecalis, insertion of Tn916 upstream of a haemolysin gene can lead to a hyper-haemolytic phenotype
(Ikeet al., 1992). The final way in which these elements can
change the DNA of the host is bytrans-acting interaction with other elements, such as that observed between Tn916 and Tn5386 (Rice et al., 2005, 2007) and mobilization of other nonconjugative plasmids and transposons leading to their transfer to new hosts (Flannagan & Clewell, 1991). The effect that a conjugative transposon has on its host is variable and extends beyond the simple provision of a new phenotype such as antibiotic resistance.
Tn916-like elements inEnterococcusspp. A clinical isolate,E. faecalisstrain DS16 (initially described
as S. faecalis ssp. zymogenes strain DS16; Tomich et al.,
1979), harbours the conjugative haemolysin-bacteriocin plasmid pAD1 (60 kb) and the nonconjugative plasmid pAD2 determining resistance to streptomycin, kanamycin and erythromycin, in addition to the chromosomally located Tn916 (Franke & Clewell, 1981). In this paper, it was shown that when strain DS16 was mated with the plasmid-freeE. faecalisstrain JH2-2, some transconjugants resistant to tetracycline contained the Tn916 determinant linked to pAD1. In addition, derivatives of DS16 devoid of pAD1 were capable of transferring tetracycline resistance to recipient strains. Transconjugants (plasmid-free) from such matings could subsequently act as donors in the transfer of
Group II intron Tn5397
orf23
orf24 orf22 orf21 orf20 orf19 orf18 orf17 orf16 orf15 orf14 orf13 orf12 tet
(M) orf6 orf9 orf10 orf7 orf8 orf5 xis-Tn int-Tn
Conjugation Resistance Regulation Recombination
oriT Tn6000 tet (S) Group II intron vap
RM systems and antirestriction genes SPIbov1 integrase Virulence-related locus(vrl) Dichelobacter nodosus Tn5397 Clostridium difficile pK214 Lactococus lactis SaPIbov2 Staphylococcus aureus tndX orf25 and orf26
Tn5801 tet (M) sav415 Tn916 hel (a) (d) (c) (b)
Fig. 1.Schematic representation of Tn916and related elements. The arrows represent the individualorfspointing in the probable direction of transcription and labelled above. The colour coding for the functionality of the predicted proteins is as follows: blue, conjugation; green, transcriptional regulation; red, accessory genes (in the case of Tn916this is tetracycline resistance); grey, insertion and excision (recombination). Genes not present in Tn916are shown in orange on the other elements. (a) Tn916: the arrow points to the position of the experimentally determinedoriT(Jaworski & Clewell, 1995). The modules are indicated by the brackets underneath and are labelled with the relevant function. (b) Tn6000: the major differences are the presence of restriction/modification genes before the conjugation module, the presence of an insertion of DNA that shares homology to thevrlof
Dichelobacter nodosusand the integrase and excisionase genes that share more homology to staphylococcal pathogenicity island genes than they do to Tn916integrase and excisionase genes. The closest homologue to various regions of DNA are indicated below the figure by brackets (adapted from Brouweret al., 2010). (c) Tn5397: the major differences are the presence oftndXin Tn5397as opposed toxisTnandintTnin Tn916, the presence of a self-splicing group II intron in Tn5397and an 88-bp deletion in the regulatory region resulting in the removal oforf12and the generation oforf25and
orf26. (d) Tn5801fromStaphylococcus aureus: the major differences are the ORFs located before the conjugation module. Most of the additional genes are hypothetical; however,sav415is predicted to encode a transposase (de Vrieset al., 2009).
861 Diversity of Gram-positive Tn916elements
another! efflux! pump! conferring! macrolide! resistance.! The!mef(A)! gene! is! carried! on! Tn1207.1,! a! 7.2kb! defective! transposon! that! corresponds! to! the! left! end! of! the! S.* pyogenes! transposon! Tn1207.3* (143).*Tn12071.1* is! only! capable! of! inserting! at! one! specific! site! within! the! pneumococcal! chromosome! and! clonal! spread! is! therefore! the! dominant! mechanism! of! dissemination! of! this! transposon! within! the! pneumococcal! population.! The! vast! majority! of! pneumococci! carrying! Tn1207.1*belong! to! the! MDR! clone,!England14H9!(144).!! ! The!mef(E)!gene!is!carried!on!the!5.4!–!5.5kb!macrolide!efflux!genetic!assembly!(mega)! element,!similar!in!sequence!to!Tn1207.1*but!lacking!the!region!upstream!of!mef*(145).*In! contrast!to!the!mef(A)!carrying!Tn1207.1,!the!mega!element!has!been!found!inserted!at! numerous!different!chromosomal!sites!in!different!serotypes!and!clonal!groups.!A!novel! composite!element!designated!Tn2009,!resulting!from!the!insertion!of!the!mega!element! into!a!Tn916!like!transposon!carrying!tet(M)!has!also!been!described!(146).!! ! A!recent!trend!in!macrolide!resistance!is!the!emergence!of!clinical!pneumococcal!strains! expressing!both!erm(B)!and!mef(E),!referred!to!as!the!‘dual!phenotype’.!This!is!the!result! of! the! dissemination! of! Tn916! derivatives! carrying! both! genes.! Analysis! of! strains! belonging!to!CC271,!a!Taiwan19FH14!type!strain!displaying!the!‘dual!phenotype,’!revealed!
erm(B)! and!mef(E)! were! contained! on! a! novel! composite! element! designated! Tn2010.!! Tn2010! is! genetically! similar! to! Tn2009,! but! with! the! addition! of! an!erm(B)! containing! element! (146).! Another! novel! composite! element! has! been! described! in! a! Hungarian! pneumococcal! isolate,! ST43,! belonging! to! CC271.! The! isolate! harboured! a! new! combination!of!Tn916,!Tn917!and!mega!that!has!been!designated!Tn2017*(147).**
!
The!novel!composite!genetic!element!harbouring!mef(I)!has!recently!been!characterised! and! is! designated! the! 5216IQ! complex.! The! 5216IQ! complex! consists! of! two! segments.! The!first!segment!is!~15.3kb!and!is!formed!from!parts!of!known!transposons,!Tn5252!and! Tn916.!The!second!segment!is!different!from!any!other!known!genetic!element!carrying! mef.!Designated!the!IQ!element,!it!ends!with!identical!transposase!genes!at!both!sides! and! contains! the!mef(I)! gene! with! an! adjacent! new!msr(D)! gene! variant! and!cat,! a! chloramphenicol!acetyltransferase!gene.!In!comparison!to!the!elements!carrying!mef(E),!
IQ! appears! to! be! nonHmobile,! and! is! therefore! scarce! amongst! macrolide! resistant! pneumococci!(148).!