The role of natural environments in the evolution of resistance traits in pathogenic bacteria Jose L. Martinez*

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1 Downloaded fom on Novembe 2, 2018 Poc.. Soc. B (2009) 276, doi: /spb Published online 8 Apil 2009 eview The ole of natual envionments in the evolution of esistance taits in pathogenic bacteia Jose L. Matinez* Depatamento de Biotecnología Micobiana, Cento Nacional de Biotecnología, Consejo Supeio de Investigaciones Científicas, Dawin 3, Cantoblanco, Madid, Spain Antibiotics ae among the most valuable compounds used fo fighting human diseases. Unfotunately, pathogenic bacteia have evolved towads esistance. One impotant and fequently fogotten aspect of antibiotics and thei esistance genes is that they evolved in non-clinical (natual) envionments befoe the use of antibiotics by humans. Given that the biosphee is mainly fomed by mico-oganisms, leaning the functional ole of antibiotics and thei esistance elements in natue has elevant implications both fo human health and fom an ecological pespective. ecent woks have suggested that some antibiotics may seve fo signalling puposes at the low concentations pobably found in natual ecosystems, wheeas some antibiotic esistance genes wee oiginally selected in thei hosts fo metabolic puposes o fo signal tafficking. Howeve, the high concentations of antibiotics eleased in specific habitats (fo instance, clinical settings) as a consequence of human activity can shift those functional oles. The pollution of natual ecosystems by antibiotics and esistance genes might have consequences fo the evolution of the micobiosphee. Wheeas antibiotics poduce tansient and usually local challenges in micobial communities, antibiotic esistance genes pesent in gene-tansfe units can spead in natue with consequences fo human health and the evolution of envionmental micobiota that ae lagely ignoed. Keywods: antibiotic esistance; envionmental mico-oganisms; antibiotic pollution; bacteial evolution; micobial ecology; infectious diseases 1. INTODUCTION Since thei intoduction fo human theapy 60 yeas ago, antibiotics have shown to be a emakable success and constitute one of the most elevant medical inventions fo educing human mobidity and motality. Unfotunately, the intensive use and misuse of antibiotics have esulted in antibiotic esistance among seveal human pathogens, educing the possibilities fo infections teatment and jeopadizing medical pocedues, such as ogan tansplantations o implants of postheses, whee infective complications ae common and antibiotic theapy is needed to pevent o teat those infections ( WHO 2000). Thee ae two main mechanisms involved in the development of antibiotic esistance, namely mutation (Matinez & Baqueo 2000) and acquisition of esistance genes (Davies 1994) by hoizontal gene tansfe (HGT). Given that human pathogens wee susceptible to antibiotics befoe the use of these dugs fo the teatment of infections, the oigin of antibiotic esistance deteminants acquied by HGT must necessaily lay in the non-pathogenic micobiosphee. In some instances, human commensals can povide antibiotic esistance to pathogens (Sibold et al. 1994). Howeve, in most cases, the antibiotic esistance genes have oiginated in the envionmental micobiota (Davies 1994, 1997). The tem antibiotic was oiginally coined to name those compounds poduced by mico-oganisms and capable *jlmtnez@cnb.csic.es of inhibiting bacteial gowth ( Waksman & Wooduff 1940), although any type of dug (natual o synthetic) used fo teating bacteial infections is fequently temed as an antibiotic nowadays. Since seveal antibiotics ae poduced by envionmental bacteia, it is conceivable that antibiotic-poducing oganisms could be the oigin of HGT-acquied antibiotic esistance genes, because these mico-oganisms must have systems to avoid the activity of the antimicobials they poduce (Benveniste & Davies 1973). Although this has been fomally demonstated in few occasions (Pang et al. 1994), we will see late that some esistance genes may have othe functional oles in thei oiginal oganisms besides antibiotic esistance (Matinez et al. 2009a). Independent of thei functional ole in non-clinical (natual) envionments, what is clea is that antibiotic esistance genes oiginated in envionmental bacteia, so that changes in natual ecosystems may impact upon antibiotic esistance and consequently human health. Among those changes, the elease of antibiotics togethe with human-linked micobiota eventually containing antibiotic esistance genes can be paticulaly impotant fo the futue evolution of antibiotic esistance in pathogenic bacteia (Baqueo et al. 2008). Howeve, this is just one side of the coin. Some mico-oganisms ae capable of poducing infections only in people who ae immunosuppessed (fo instance, those with AIDS, unde chemotheapy o afte tansplantation), debilitated eceived 24 Febuay 2009 Accepted 16 Mach This jounal is q 2009 The oyal Society

2 Downloaded fom on Novembe 2, J. L. Matinez eview. Antibiotic esistance in natue o with a basal disease. These oganisms, which do not usually infect healthy people, ae consideed as oppotunistic pathogens (Quinn 1998; Gaynes & Edwads 2005). In some instances, human commensal bacteia poduce oppotunistic infections. Nevetheless, some envionmental bacteia ae also pominent oppotunistic pathogens. One of the most poblematic chaacteistics of oppotunistic pathogens with an envionmental oigin is that they usually display low susceptibility to antibiotics (Hancock 1998; Quinn 1998; Hancock & Speet 2000; Feaa 2006; Beidenstein et al. 2008; Matinez et al. 2009b). This is a common chaacteistic of all membes of a given bacteial species. Fo instance, all the stains of Pseudomonas aeuginosa contain the same chomosomally encoded multidug (MD) efflux pumps (Alonso et al. 1999) and ampc beta-lactamase genes that contibute to the intinsic phenotype of esistance displayed by this bacteial species (Bonomo & Szabo 2006). This indicates that those esistance deteminants ae ancient elements pesent in the chomosomes of all membes of each bacteial species befoe its subspecific divesification some hundeds of thousands of yeas ago. This is consistent with the concept that the phenotype of intinsic esistance to antibiotics was acquied in natual envionments, outside clinical wads, long ago and is not the esult of ecent antibiotic usage fo human theapy o faming. As stated fo efflux pumps, these elements have pedated the antibiotic ea and thei natual ole is unlikely to be elated to antibiotic use. Fo instance, synthetic antibiotics such as quinolones ae unlikely to have selected fo efflux pumps evolution (Piddock 2006a). All these featues justify the concept that natual envionments ae impotant fo the evolution of antibiotic esistance in bacteial pathogens. To undestand which mechanisms have been and ae cuently being involved in this evolution, we will eview the ecological oles of antibiotic and esistance genes in natual ecosystems and consequently which ae the foces that shaped the evolution of esistance deteminants befoe human use of antibiotics. We will eview as well how bacteial pathogens acquie esistance fom envionmental bacteial and finally we will discuss the consequences of the elease of antibiotics and esistance genes in natual ecosystems fo the cuent and futue evolution of esistance in bacteial pathogens. 2. FUNCTIONS OF ANTIBIOTICS AND ANTIBIOTIC ESISTANCE GENES IN NATUAL ECOSYSTEMS Within this eview, we will dub as natual ecosystems o envionmental bacteia those that do not have a humanassociated (commensal o infective) evolutionay linkage. Although natual ecosystems can suffe antibiotic pollution as the consequence of human activity, envionmental bacteia ae not globally unde the stong antibiotic selective pessue suffeed by human pathogens, which ae challenged with antibiotics duing theapy. One impotant featue to popely analyse antibiotic esistance in natual ecosystems is to undestand the function of antibiotics and thei esistance elements in those habitats. Since seveal of the antibiotics used fo teating infections ae synthesized by soil mico-oganisms, it has been assumed that the function of these compounds in natue should be to inhibit the gowth of the micobial competitos of the antibiotic poduces ( Waksman & Wooduff 1940). Convesely, it has been poposed that antibiotic esistance deteminants have specifically evolved to avoid the activity of antibiotics, being the antibiotic poduces a majo souce of esistance genes (Benveniste & Davies 1973). Wheeas in some occasions, these weapon/shield oles ae easonable explanations fo the functions in natue of, espectively, antibiotics and esistance deteminants (de Loenzo et al. 1984; Pang et al. 1994), some othe oles have been poposed fo them. It has been discussed that some antibiotics may seve fo signalling puposes, at the low concentations pobably encounteed in most natual envionments (Davies 2006; Yim et al. 2006b, 2007; Fajado & Matinez 2008). This poposal is based on expeimental wok showing that low concentations of antibiotics tigge specific tansciptional changes, which ae independent of the geneal stess esponse micobial netwoks (Goh et al. 2002; Tsui et al. 2004; Linaes et al. 2006; Yim et al. 2006a). Inasmuch, some compounds peviously chaacteized as bona fide signal molecules also have antimicobial activity (Ji et al. 1997; Deziel et al. 2004; Kaufmann et al. 2005), futhe suppoting the dual ole of these compounds. Altogethe, these studies indicate that the pimay function of some antibiotics might be intecellula signalling in natual ecosystems, being inhibitos of bacteial gowth only at the high concentations used fo theapy. Concening antibiotic esistance genes, some of them, cuently pesent in pathogenic bacteia, ae involved in antibiotic detoxification in the poduce oganisms (Pang et al. 1994) o in the esistance to toxic compounds poduced by plants o by thei associated micobiota (Bais et al. 2005, 2006), a featue that fits well with thei ole as shields. Howeve, avoiding antibiotic activity is not always the pimay ole of some well-known antibiotic esistance deteminants in natual ecosystems. Fo instance, it has been suggested that plasmid-encoded beta-lactamases, which ae vey poficient antibiotic esistance deteminants acquied by pathogenic bacteia though HGT, might oiginally have been penicillin-binding poteins involved in the synthesis of peptidoglycan, and thei activity against beta-lactams is a side effect of thei oiginal function (Kelly et al. 1986; Massova & Mobashey 1998; Meoueh et al. 2003). Similaly, the chomosomal 2 0 -N-acetyltansfease of Povidencia stuatii, an enzyme involved in the modification of the bacteial peptidoglycan, can inactivate gentamycin and is consideed as an antibiotic esistance deteminant, although its oiginal pimay function is a diffeent one ( Macinga & athe 1999). These examples illustate the concept that a deteminant which contibutes to the esistance of human pathogens to antibiotics can be involved in cental metabolic pocesses of envionmental bacteia in thei natual habitats. As shown hee, a metabolic enzyme with a substate pesenting stuctual similaities to a given antibiotic might modify this antibiotic, thus seving as an antibiotic esistance gene in envionments with a high antibiotic load, such as clinical settings. Othe mechanisms that do not involve antibiotic degadation wee pobably selected as well in natue to play a pimay functional ole diffeent fom antibiotic esistance. This is exemplified by the esistance to quinolones, a synthetic family of antibiotics intoduced Poc.. Soc. B (2009)

3 Downloaded fom on Novembe 2, 2018 eview. Antibiotic esistance in natue J. L. Matinez 2523 fo theapy in the 1960s. In spite of thei synthetic oigin, quinolones ae a favouite substate of MD efflux pumps, and envionmental bacteia, isolated befoe the invention of quinolones, can efflux these dugs (Alonso et al. 1999), indicating that esistance is not the pimay function of those deteminants. A simila situation might happen with Qn, the fist plasmid-encoded quinolone esistance deteminant (Matinez-Matinez et al. 1998). It has been shown that qn genes ae chomosomally encoded in watebone bacteia (Poiel et al. 2005; Sanchez et al. 2008). Futhemoe, the high consevation in the synteny of the egions suounding these genes togethe with the lack of elements associated to tansposition o insetion events in thei vicinity (Sanchez et al. 2008) suppots the notion that these deteminants oiginated in watebone bacteia (Poiel et al. 2005), in habitats whee the pesence of quinolones is not expected. As in the case of antibiotics, which can have a pimay ole diffeent fom killing competitos, we can then conclude that the pimay functions in natual ecosystems of some esistance genes (e.g. metabolic enzymes o efflux pumps involved in signal tafficking) ae not to avoid the activity of antibiotics. 3. INTINSIC ESISTANCE Antibiotic esistance has been mainly consideed as an evolutionay pocess diven by antibiotics selective pessue: bacteia ae challenged with antibiotics and develop esistance as the consequence of mutation (Matinez & Baqueo 2000) o HGT (Davies 1994). In fact, given the time equied fo the evolution of Metazoans ( Navas et al. 2007), emegence and spead of esistance is consideed as one of the few evolutionay pocesses that can be studied in eal time (Salmond & Welch 2008). In spite of these consideations, the ecent use (in evolutionay tems) of antibiotics fo human theapy and faming is not the unique foce diving evolution towads antibiotic esistance of human pathogens. Some bacteial species, pesenting intinsic low susceptibilities to antibiotics, have an envionmental oigin in habitats whee thee is not a high antibiotic load. Futhemoe, those esponsible fo infections in hospitals, such as P. aeuginosa, Acinetobacte baumannii o Stenotophomonas maltophilia (Quinn 1998), ae not themselves poduces of classical antibiotics and thus do not need to cay esistance genes in thei genomes. A naive explanation to justify this esistance should be based on the fact that the taget oganisms used in the sceening of antibiotics wee the classical human pathogens, so that it would not be ae that envionmental bacteia ae able to esist those compounds. Howeve, seveal antibiotics ae boad spectum, and thei tagets ae pesent in envionmental bacteia as well as in human pathogens. An altenative hypothesis emeges if we conside antibiotics as egula metabolites that have othe functions besides killing competitos as discussed above. Fee-living bacteia such as those afoementioned have lage genomes that allow them to colonize diffeent envionments (Cases et al. 2003) and on occasion possess a lage numbe of biodegadative enzymes, which may coopeate in the degadation and use of antibiotics as food esouce (Dantas et al. 2008). Besides, they fom complex communities that equie intecellula communication, and the mechanism fo signal tafficking can be involved in esistance if the mechanisms fo tanspoting the signal outside bacteia ae co-opted by a given antibiotic. This is the case of MD efflux pumps (Matinez et al. 2009b), a family of tanspotes that ae pesent in all oganisms (Saie & Paulsen 2001; Piddock 2006a; Lubelski et al. 2007), with the same chomosomally encoded MD pumps being pesent in all stains of a given species (Alonso et al. 1999; Alonso & Matinez 2001; Sanchez et al. 2004). It has been shown that besides intinsic antibiotic esistance and esistance against othe toxic compounds (Silve & Phung 1996; Henandez et al. 1998; amos et al. 2002; Sanchez et al. 2005), MD pumps can contibute to viulence ( Piddock 2006b) and ae capable of efflux of intecellula signal compounds ( Evans et al. 1998; Kohle et al. 2001). Finally, intinsically esistant envionmental bacteia can colonize the hizosphee, a habitat that might contain toxic compounds poduced by eithe the plants o the associated micobiota (Matilla et al. 2007). The lage metabolic vesatility of fee-living pathogens, which allows the colonization of such divese habitats (Cases et al. 2003), pobably allows them also to esist the toxicity of seveal compounds, including antibiotics (Gonzalez- Pasayo & Matinez-omeo 2000; Matilla et al. 2007; Matinez et al. 2009b). The complexity of the mechanisms involved in intinsic esistance is futhe exemplified by studies showing that a lage numbe of genes, belonging to diffeent functional categoies, contibute to intinsic esistance to antibiotics (Beidenstein et al. 2008; Fajado et al. 2008; Tamae et al. 2008). All these consideations justify the conclusion that the phenotype of intinsic esistance displayed by some oppotunistic pathogens with an envionmental oigin has been acquied, duing the evolution of these micooganisms in thei natual habitats, long befoe human discovey of antibiotics. 4. ACQUIED ESISTANCE, AN EVOLUTIONAY PESPECTIVE In contast to intinsic esistance, which has been developed by bacteial populations befoe human use of antimicobials, acquied antibiotic esistance is a ecent event in the evolution of human pathogens in which the main selective foce has been the human use of antibiotics (Levy 1998; Levy & Mashall 2004; Alekshun & Levy 2007; Salmond & Welch 2008). This view was ealie suppoted by the studies of Naomi Datta showing that the families of plasmids pesent in enteobacteia befoe and afte the use of antibiotics ae the same, and the only diffeence is that those plasmids have acquied esistance genes afte antibiotics wee intoduced fo theapy (Datta & Hughes 1983). As stated above, antibiotic esistance can be acquied eithe by mutation o by HGT. Wheeas mutation-diven esistance can happen duing antibiotic teatment, HGT-acquied esistance equies a dono of the esistance genes, so that the fist tansfe event equies contact between an envionmental mico-oganism (o its DNA in the case of tansfomation) and a humanassociated bacteia. Although we will mainly efe to the acquisition of esistance by bacteial pathogens, it is woth emembeing that commensal bacteia can seve as vectos fo the tansmission of esistance genes between Poc.. Soc. B (2009)

4 Downloaded fom on Novembe 2, J. L. Matinez eview. Antibiotic esistance in natue Figue 1. Ecological landscapes in antibiotic esistance. The deteminants that can contibute to antibiotic esistance in pathogenic bacteia ae distibuted in all types of envionments. Howeve, neithe thei functional ole no the antibiotic selective pessue is the same in all of these envionments. In natual (non-clinical) ecosystems (white squae), antibiotic concentations ae usually low. Some deteminants () can seve fo antibiotics detoxification in micobial poduces o to avoid the inhibitoy activities of antibiotics at places in which the concentations of these dugs ae locally high. Howeve, some othes (), which can contibute to esistance in habitats with a high antibiotic load, ae involved in metabolic o signalling pocessing in thei oiginal hosts. Opposite to this situation, the main functional ole of these elements in habitats with a high antibiotic selective pessue such as clinical settings (dak gey squae) will be esistance. Non-clinical envionments eceiving wastes containing antibiotics and human-linked (o animal-linked in the case of fams) bacteia (gey squae) will be eniched in genetic platfoms containing esistance genes aleady selected in bacteial pathogens, whose function is just esistance (see text fo moe details). envionmental and pathogenic mico-oganisms. This situation implies the existence of thee diffeent landscapes impotant in the evolution of esistance of human pathogens (figue 1). The fist level is the whole micobiosphee. Envionmental bacteia contain a lage numbe of genes capable of confeing antibiotic esistance to human pathogens upon thei tansfe though HGT (D Acosta et al. 2006; Matinez et al. 2007; Wight 2007). As discussed above, some of these deteminants have been selected eithe fo detoxification puposes in antibiotic poduces, o to avoid the activity of plants o mico-oganisms synthesized antimicobials, wheeas othes have evolved to play functions diffeent fom antibiotic esistance. In any case, stong selective pessue by human-poduced antibiotics did not have any elevance in the pimay evolution of these deteminants in natual ecosystems. Nevetheless, the elease of high amounts of antibiotics and thei esistance genes as a consequence of human activities in natual ecosystems has changed this situation in the last decades (see below). The second level consists of those habitats with fequent contact of human-associated bacteia with the envionmental micobiota, fo instance wastewates. Since HGT equies the two patnes to be pesent in the same place, these allocations will be hotspots fo the fist step in the acquisition of antibiotic esistance genes by pathogenic bacteia: thei tansfe fom the envionmental bacteia to the human-associated ones. Futhemoe, humanassociated wastewates fequently contain antibiotics and esistance genes, which incease the pobabilities fo DNA exchange and fo the selection of esistance (Baqueo et al. 2008). The thid level is the teated patient itself (o the animal in the case of antibiotics used fo faming puposes). DNA tansfe fom commensals to pathogenic bacteia (fo instance, in the development of esistance to beta-lactams by Gam-positive bacteia (Spatt et al. 1992; Sibold et al. 1994; eichmann et al. 1997)) and between pathogenic bacteia in the case of polymicobial infections has been descibed (Matinez-Suaez et al. 1987). Howeve, the most impotant mechanism fo developing esistance duing infections is pobably mutation ( Macia et al. 2005). Actually, mutants with highe mutation ates than those of the wild-type stains ae selected in clinical habitats (LeClec et al. 1996; Olive et al. 2000; Baqueo et al. 2004) pobably by a second-ode selective pocess, which favou thei evolution towads esistance in those envionments with a stong antibiotic selective pessue (Macia et al. 2005). Unde teatment, it can be thus pedicted that acquisition of esistance mutations and mutation-diven divesification of genes, aleady acquied by HGT, will be the most elevant evolutionay pathways of bacteial pathogens. Since the antibiotic selective pessue is diffeent in these thee envionments, we can conclude that the elevance of the function of these elements as esistance deteminants is diffeent as well. In the fist one (natual ecosystems), antibiotic concentations ae not globally high, although they might be locally elevant, and the main function of those deteminants would not necessay be esistance. Contaily, in the teated host, with a high antibiotic load, HGT-acquied esistance deteminants do not have eithe the genetic egulatoy netwok no the biochemical patnes (substates, othe enzymes fom the same metabolic pathway) they have in thei oiginal host, and thei unique function will be just esistance (Matinez 2008), in an example of co-optive evolution, o exaptation (Gould & Lloyd 1999). The evolutionay tajectoy of an antibiotic esistance gene fom an envionmental mico-oganism to become a bona fide esistance deteminant in pathogenic bacteia pesents two bottlenecks. The fist one consists of its tansfe and establishment in the human-associated micobiota. The use of cultue-based (D Acosta et al. 2006; Wight 2007) and metagenomics ( Handelsman 2004; Moi et al. 2008; Allen et al. 2009a,b) methods to analyse envionmental micobiota has demonstated that the numbe of potential esistance deteminants in envionmental bacteia is huge. Howeve, vey few of them ae cuently pesent in human pathogens ( Matinez et al. 2007). Fou elements contibute to this situation. Fist, only those genes that can coexist with human pathogens ae suitable to contibute to esistance. Fo instance, it is vey unlikely that esistance genes found in oganisms fom the deep teestial subsuface (Bown & Balkwill 2008), o pesent in the deep Geenland ice coe (Miteva et al. 2004), might contibute to esistance of bacteial pathogens. Second, only those genes ecuited by Poc.. Soc. B (2009)

5 Downloaded fom on Novembe 2, 2018 eview. Antibiotic esistance in natue J. L. Matinez 2525 pesistance of esistant bacteia a time Figue 2. Effect of antibiotic esistance on bacteial fitness. The acquisition of a phenotype of esistance is fequently linked to a fitness cost, which esults in esistant populations being outcompeted by susceptible ones in the absence of antibiotics (a). Howeve, some antibiotic esistance mutations have no cost (b) and esistant bacteia ae maintained, o ae even beneficial by inceasing bacteial fitness (c), in which case the esistant bacteia pevail ove susceptible ones even in the absence of antibiotics. Futhemoe, esistant bacteia can acquie compensatoy mutations that incease thei fitness to the level of the susceptible population (d), so that these compensated mutants ae maintained, o eventually incease thei fitness (e) and the compensated mutants outcompete the susceptible bacteia even in the absence of antibiotics. gene-tansfe systems compatible with human pathogens will be tansfeed. The tansfe of these systems is limited by baies that include thei host ange (e.g. the hosts in which the plasmids can eplicate), the exclusion between diffeent plasmids and the DNA estiction/modification systems, among othes (see Thomas & Nielsen (2005) fo a eview in depth of those mechanisms). Diect tansfomation can be also impotant fo natually competent pathogens. In this case, the maintenance of the tansfeed DNA equies its integation in the ecipient chomosome. The pobability of this ecombination event vaies as a function of the oganism (e.g. 0.1% of the intenalized fagments ae integated in Acinetobacte baylyi, wheeas moe than 25% of them ae successfully ecombined in Bacillus subtilis and Steptococcus pneumoniae ( Thomas & Nielsen 2005)) and the homology between the incoming DNA and the egions of the chomosome of the ecipient cell involved in ecombination (Lovett et al. 2002; Thomas & Nielsen 2005). Mutato stains, which display a high mutation ate, ae also highly ecombinogenic (Matic et al. 2000). Thid, those elements that poduce stong fitness costs in thei hosts (figue 2) will be counteselected (Andesson & Levin 1999; Moosini et al. 2000), unless compensatoy mutations ae easily selectable (Bjokman et al. 2000; Paulande et al. 2007; Lofmak et al. 2008). Fouth fo those elements that fulfil the thee fome citeia a founde effect is expected (Matinez et al. 2009a), in such a way that the fist esistance deteminant acquied by HGT will spead and the pobabilities fo the acquisition of a new one will be low, unless antibiotic selective pessue changes. e c d b The second bottleneck consists of the stong selective pessue (mainly duing antibiotic theapy) suffeed by pathogenic bacteia. This selective pessue can lead to the divesification of the HGT-acquied genes. TEM betalactamases ae a good example of this pocess. TEM-1, the fist membe of this family of enzymes, inactivates the fist class of beta-lactams and has been pevalent fo decades in esistant enteic Gam-negative bacilli ( oy et al. 1983). Afte the intoduction of beta-lactamase inhibitos and new geneations of beta-lactams, a stong divesification in TEM-1 deived enzymes has occued (Pateson & Bonomo 2005) to the point that the numbe of mutant TEM enzymes ae now above one hunded. A simila situation is cuently occuing with othe beta-lactamases such as those belonging to the CTX-M family. The finding of two CTX-M deivatives suounded by identical sequences in the same IncN plasmids indicates that the evolution of these enzymes has occued afte thei integation in those plasmids ( Novais et al. 2007). These examples suppot the conclusion that those new (evolved) esistance genes wee not pesent befoe in natue and thei emegence is a diect consequence of the evolutionay pocess tiggeed by human use of antibiotics. Whethe o not thei eintoduction in natual ecosystems may challenge the envionmental micobiota is a topic that has not been popely addessed. 5. CHANGES IN NATUAL ECOSYSTEMS AND FUTUE EVOLUTION OF ANTIBIOTIC ESISTANCE Since antibiotic esistance genes oiginated in envionmental bacteia, an impotant topic to addess would be to state whethe changes in the natual ecosystems, mainly as the consequences of human activities, can challenge the envionmental micobiota in such a way that these modifications alte the esistance of human pathogens (Alonso et al. 2001). Antibiotics ae nowadays used, not just fo human theapy, but also fo faming puposes. This includes the teatment of infections, and thei use fo pomoting faste gowth of livestock ( Febe 2003). Although the usage of antibiotics fo this last pupose has been banned in some counties, the elease of antibiotics that ae not used fo peventing o teating human infections is still vey high. Togethe with the antibiotics used in clinics, this has meant the contamination with antibiotics at fams, ives that eceive wastewates and lands eceiving antibioticcontaminated manue occus fequently (Cabello 2006). This can acceleate evolution towads esistance and inceases the isks fo its tansfe to human pathogens, which can be pesent as well in these ecosystems (Baqueo et al. 2008). Fo instance, the pesence of plasmid-encoded qn genes in envionmental Aeomonas spp. has been ecently epoted (Cattoi et al. 2008). Since these deteminants ae chomosomally encoded in watebone bacteia, thei integation in a gene-tansfe element, may be as the consequence of the use of quinolones in aquacultue and could be a fist step fo its tansfe to human pathogens. Notably, the same plasmid containing the same qn gene has been found in geogaphically distant allocations (Picao et al. 2008), indicating that once antibiotic esistance genes ae integated in gene-tansfe elements, they then have a bette chance of dissemination and to eventually emain in bacteial populations. Poc.. Soc. B (2009)

6 Downloaded fom on Novembe 2, J. L. Matinez eview. Antibiotic esistance in natue Anothe type of contamination that can be elevant fo the evolution of esistance in bacteial pathogens is constituted by the antibiotic esistance genes themselves. The finding of esistant oganisms woldwide should not be taken as a supise, given the envionmental oigin of esistance genes ( Davies 1994). What is poblematic howeve is the finding of esistance deteminants, aleady well established in bacteial pathogens, in envionments without a histoy of antibiotic contamination (Pallecchi et al. 2008). Fo instance, emote human populations without known antibiotic exposue cay antibiotic esistant bacteia (Genet et al. 2004; Batoloni et al. 2009) and esistant bacteia ae also established in wild animal populations, despite not eceiving any antibiotic (Gillive et al. 1999; Livemoe et al.2001). The pesence of the same antibiotic esistance genes, associated with the same genetic platfoms (integons, tansposons and plasmids) in both bacteial pathogens and pistine envionments with non-detectable concentations of antibiotics, implies the existence of a woldwide-distibuted antibiotic esistance backgound. These esistance deteminants ae aleady pesent in the envionment in genetic platfoms compatible with bacteial pathogens and thus eady to be tansfeed by HGT unde antibiotic selection. This suggests that some antibiotic esistance genes ae difficult to eliminate even in the absence of antibiotic selective pessue, and thus emain in the envionment modifying the genetic epetoie and eventually the population dynamics of envionmental mico-oganisms. Thee ae diffeent easons fo explaining the maintenance of esistance-tansfe units in the absence of selective pessue. Some esistance mechanisms have no cost fo the bacteia, o even ende a highe fitness in specific envionments (Alonso et al. 2004; Luo et al. 2005; Balsalobe & de la Campa 2008), in such a way that esistant mico-oganisms ae not outcompeted by thei wild-type countepats (figue 2). Even fo elevant fitness costs, compensatoy mutations educing bacteial buden might be selected (Bjokman et al. 2000; Paulande et al. 2007; Lofmak et al. 2008). Seveal plasmids encode toxin antitoxin systems ( Jaffe et al. 1985; Gedes et al. 1986; Hiaga et al. 1986; Hayes 2003). If those plasmids contain antibiotic esistance genes (Moitz & Hegenothe 2007; Peichon et al. 2008; Sletvold et al. 2008), the pobability fo the maintenance of esistance might be high (figue 3). Antibiotic esistance genes can be clusteed in the same unit (fo instance an integon (Stokes & Hall 1989; Mazel 2006)) with othe elements that can confe an ecological advantage (figue 3). This includes esistance to toxic compounds (e.g. antibiotics, heavy metals, biocides) and othe elements, such as sideophoes, micocins o toxins, which can favou the suvival of bacteia in a given habitat (Delgado-Iibaen et al. 1987; Matinez & Peez-Diaz 1990; Heeo et al. 2008). Unde these cicumstances, esistance can be co-selected by its associated tait in the absence of antibiotics. Since heavy metal esistance and biocide esistance genes can be associated with antibiotic esistance, this implies that heavy metal contamination, as well as the usage of biocides, might select fo antibiotic esistance in natual envionments ( Novick & oth 1968; Foste 1983; Moozzi et al. 1986; Belliveau T A P a T A P T A P b e et al. 1991; Summes et al. 1993; Dhakephalka & Chopade 1994; Henandez et al. 1998; McAthu & Tuckfield 2000; Gaze et al. 2005; Sanchez et al. 2005; Stepanauskas et al. 2006). Most woks on the effects of human activity on the biosphee ae based on the study of highe oganisms. Howeve, the majoity of life is micobial, and the effects of envionmental changes on the micobiosphee ae lagely ignoed. We know that pollution by antibiotics and antibiotic esistance genes can alte the envionmental micobiota. Nevetheless, we ignoe whethe pat of these alteations might emain ove the long tem. Wheeas antibiotics ae degaded in natue, the genetic platfoms containing esistance genes ae auto-eplicative elements that might be athe stable. Seveal antibiotics and esistance genes have an envionmental oigin in habitats whee they have evolved without contact with humans fo hundeds of thousands of yeas. On occasions, the selective foce fo the evolution of esistance genes in T A b b e Figue 3. Maintenance of antibiotic esistance platfoms in the absence of antibiotic selective pessue. Antibiotic esistance genes ae acquied by genetic platfoms that can disseminate among bacteial populations. The Figue epesents a bacteium caying one plasmid. The plasmid can contain diffeent genes, which encode deteminants with adaptive values fo the ecipient bacteium. This includes antibiotic esistance genes (), biocide esistance o heavy metal esistance deteminants (b) o ecologically ewading elements (e) such as sideophoes, micocins o toxins among othes. Even in the absence of antibiotics, the pesence of these deteminants associated in the same plasmid can favou its selection and thus co-selection of antibiotic esistance. On the othe hand, seveal plasmids expess toxin ( T)/antitoxin (A) systems that impede thei loss. The antitoxin binds the toxin and thus impedes bacteial death. Howeve, if the plasmid is lost (a), the poduction of those two poteins is stopped and the antitoxin is apidly degaded by a potease ( P), libeating the toxin and allowing bacteial killing. If the plasmid is maintained ( b), the antitoxin emains to be continuously poduced and the activity of the toxin is inhibited. If this type of plasmids contains esistance genes, thei loss, even in the absence of antibiotic selective pessue, is unlikely. T A Poc.. Soc. B (2009)

7 Downloaded fom on Novembe 2, 2018 eview. Antibiotic esistance in natue J. L. Matinez 2527 natual envionments has been the pesence of a given toxic compound (not necessaily an antibiotic). Howeve, at othe times, the deteminants that nowadays contibute to esistance of bacteial pathogens wee selected fo metabolic, stuctual o signal-tafficking puposes (see above) in habitats with low antibiotic selective pessue. The intoduction by humans of antibiotics fo theapy has changed this situation in habitats whee the main selective foce is now the pesence of high concentations of antibiotics. The enichment, in populations of envionmental bacteia, of genetic platfoms containing antibiotic esistance genes as the consequence of this vey ecent (in evolutionay tems) human-linked antibiotic use, is acceleating the evolution and spead of esistance among micobial populations, including bacteial pathogens. 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