Transfer of Antibiotic-Resistance Genes Via Phage-Related Mobile Elements Maryury Brown-Jaque, William Calero-Cáceres, Maite Muniesa *

Plasmid 79 (2015) 1–7

Contents lists available at ScienceDirect

Plasmid

journal homepage: www.elsevier.com/locate/yplas

Review Transfer of antibiotic-resistance genes via phage-related mobile elements Maryury Brown-Jaque, William Calero-Cáceres, Maite Muniesa *

Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain

ARTICLE INFO ABSTRACT

Article history: Antibiotic resistance is a major concern for society because it threatens the effective pre- Received 10 December 2014 vention of infectious diseases. While some bacterial strains display intrinsic resistance, others Accepted 13 January 2015 achieve antibiotic resistance by mutation, by the recombination of foreign DNA into the Available online 15 January 2015 chromosome or by horizontal gene acquisition. In many cases, these three mechanisms Communicated by Saleem Khan operate together. Several mobile genetic elements (MGEs) have been reported to mobilize different types of resistance genes and despite sharing common features, they are often Keywords: considered and studied separately. Bacteriophages and phage-related particles have re- Bacteriophages MGE cently been highlighted as MGEs that transfer antibiotic resistance. This review focuses on Antibiotic resistance phages, phage-related elements and on composite MGEs (phages-MGEs) involved in an- ICE tibiotic resistance mobility. We review common features of these elements, rather than GTA differences, and provide a broad overview of the antibiotic resistance transfer mechanisms observed in nature, which is a necessary ﬁrst step to controlling them. © 2015 Elsevier Inc. All rights reserved.

Contents

1. Introduction ...... 1 2. Classic elements involved in ARG transfer ...... 2 2.1. MGEs that mediate ARG transfer with cell–cell contact ...... 2 2.2. MGEs that mediate ARG transfer without cell–cell contact ...... 4 3. MGEs composed by phages or phage-related elements ...... 5 3.1. Plasmid-phage ...... 5 3.2. Transposon-phage ...... 5 3.3. GI-phage ...... 5 3.4. Gene transfer agents (GTA) ...... 5 4. Concluding remarks ...... 6

Acknowledgments ...... 6 References ...... 6

1. Introduction

Bacterial genomes exhibit dynamic plasticity and are shaped by horizontal gene transfer (HGT), genome re- arrangement, and the activities of mobile DNA elements * Corresponding author. Department of Microbiology, Faculty of Biology, Av. Diagonal 643, 08028 Barcelona, Spain. Fax: +34 3 4039047. (Darmon and Leach, 2014). The number of reports E-mail address: [email protected] (M. Muniesa). of HGT mechanisms between distinct organisms has http://dx.doi.org/10.1016/j.plasmid.2015.01.001 0147-619X/© 2015 Elsevier Inc. All rights reserved. 2 M. Brown-Jaque et al./Plasmid 79 (2015) 1–7 increased substantially in the recent years, not only for bac- Many of the elements that we present in this section teria, but also for eukaryotic cells (Keeling and Palmer, 2008). share common features. There is a group that replicates in- HGT vectors suffer a dual evolutionary pressure on their sur- dependently of the host chromosome while there are some vival: via vertical gene transfer (VGT) from parenteral strains that could become integrated into bacteria. Among the latter, to their progeny and by HGT via, at least, the three well the most common trait is the presence of integrases: the known mechanisms: conjugation, transformation and trans- enzymes that allow insertion of the element into the host duction. Some of these mechanisms are mediated by mobile chromosome. Many integrases are members of the tyro- genetic elements (MGEs) (Gogarten and Townsend, 2005). sine recombinase family (Esposito and Scocca, 1997). Many Many MGEs are able to capture bacterial DNA and move MGEs, such as phages, plasmids, genomic islands or inte- it between hosts. The collection of genes present in one grative conjugative elements (ICEs) (Boyd et al., 2009; species can then potentially be transferred to many others Casjens, 2003; Muniesa et al., 2006; Wozniak and Waldor, (Bushman, 2002). Horizontal mobilization broadens the gene 2010), provide the required integrase genes, while some repertoire available to organisms, improving their chances MGEs hijack integrase genes located in the host bacterial to evolve. Bacterial genes may be randomly recruited from chromosomes (Das et al., 2013). Many of these elements can the original host into the recipient cell, but they are selec- revert from their integrated state and excise themselves from tively kept only if they confer advantage on the new host the chromosome similarly to how temperate phages do. In strain through a marked impact on its fitness. Mecha- the extracellular state, they are susceptible to movement nisms of HGT are responsible of phenotypic variability in between cells. The horizontal mobility mechanisms will metabolism, virulence and antibiotic resistance. depend on the proximity of the donor cell to the recipient. Bacteria have developed mechanisms of resistance that combat the threat of different antibiotics produced by com- 2.1. MGEs that mediate ARG transfer with cell–cell contact petitor microorganisms. While some bacterial strains display intrinsic resistance (Davies and Davies, 2010), in others an- The most evident difference between the elements that tibiotic resistance is acquired by mutations in different mobilize ARGs is the requirement for cell-to-cell contact, chromosomal loci, by the recombination of foreign DNA into or lack thereof. The mechanism that requires contact is con- the chromosome or by horizontal acquisition of antibiotic jugation, which operates in the transfer of plasmids, resistance genes (ARGs) (Davies, 1997). In many cases, these transposons, integrons, and other related elements (Fig. 1). three mechanisms operate together. For example, the re- Plasmids are elements that have been widely explored sistance to a given antibiotic acquired by mutation of the as the purveyors of antibiotic resistance mobility and have antibiotic target gene could later be transferred laterally by been found to allow for the rapid development of multi- an R-plasmid or a temperate phage. DNA fragments encoded ple antibiotic resistance. Plasmids are capable of replicating in an MGE can recombine either with the bacterial chro- together with the cell as self-replicating elements and to mosome or with other elements present in bacterial cells. transfer resistance vertically, or to be mobilized horizon- In addition, many MGEs have the capacity to insert them- tally by incorporation into a conjugative plasmid. ARGs can selves into the bacterial chromosome, resulting in be conjugally transferred to cells from a broad range of or- chromosomally-encoded resistance. This insertion could be ganisms; in Gram-negative bacteria the process is mediated either reversible or irreversible, depending on the element. by the type IV secretion system (T4SS) (Cabezón et al., 2014). If irreversible, the element loses its intrinsic horizontal mo- Some plasmids can also be inserted into the bacterial chro- bility, while other MGEs always remain as independent mosome (Boccard et al., 1989) and mobilized vertically. replicons and can be horizontally transferred. Because of the Chromosomal resistance can also often be captured in clear advantages provided by the incorporation of ARGs, a transposon (Chancey et al., 2012; Quintiliani and Courvalin, there is a variety of HGT mechanisms that have generated 1996). The way in which transposons are integrated into and a considerable range of elements. Such diversity leads to the excised from the chromosome depends on whether they are need for continuous adaptation and classification and to the “autonomous” or “non-autonomous”. Autonomous generation of new terms and definitions of the intermedi- transposons move by themselves; while non-autonomous ate elements. transposons require the presence of other transposons to In this review, we focus on composites within gene trans- move. Integration is dependent on the presence of a fer pathways that result in intermediate or composite transposase that recognizes direct repeats, but the main elements that mobilize ARGs, and particularly, on those in- point is that transposons per se are not transferred hori- termediates that involve phages or phage-derived particles zontally. Transposons could be simple or composed. Simple that transfer ARGs. transposons are in fact insertion sequences (IS), that do not possess ARGs, but can contribute to variations in antibiotic resistance by moderating the expression of contiguous 2. Classic elements involved in ARG transfer genes (i.e. introducing strong promoters), mobilizing contiguous genes or simply inactivating genes. IS are flanked A common trend in science is to organize reality into clus- between short, inverted, repeated sequences that flank its ters, sections, types and so forth, with the intention of gene coding region. Composite transposons, in compari- providing some order and structure that could help us to son to simple transposons have genes, often ARGs, flanked understand the diversity we find in the world. However, by two separate and not always identical IS elements. The sometimes the specificity of the classifications prevents entire fragment of DNA spanning from one IS element to wider observations of the big picture. the other is transposed as one complete unit. M. Brown-Jaque et al./Plasmid 79 (2015) 1–7 3

Donor cell Receptor cell

Requires cell-cell contact ARGs mobilization in: ICE GI - Plasmids Integron - Integrons/ Transposons & Tn - GI (through conjugation) - ICE (through conjugation)

plasmid

Phage particles Specialized transduction Not cell-cell contact ARGs mobilization in: prophage -Phages GI Integron - Composed elements GTAs & Tn Plasmid-phage Transposon-phage Generalized GI-phage transduction -GTAs

plasmid

Fig. 1. Schematic representation of MGEs responsible for antibiotic resistance mobility distributed by their requirement of close cell-to-cell contact for a successful ARG transfer. ARG: antibiotic resistance gene; GI: genomic island; ICE: integrative conjugative elements, GTA: gene transfer agent; Tn: transposon.

Integrons (Cambray et al., 2010) are similar to Some GIs are mobile and others are not, or no longer mobile transposons to some extent; they may also harbor antibi- (Boyd et al., 2009; Juhas et al., 2009). A number of GIs are otic resistance and the main difference is that the enzyme capable of integration into the host chromosome, exci- that allows their integration is an integrase instead of a sion, and transfer to a new host by transformation, transposase. Integrons are not MGEs in themselves. Rather, conjugation or transduction. Examples of GIs that encode their gene cassettes could be integrated or split from the ARGs can be found in Staphylococcus aureus (Katayama et al., integron mediated by the integrase. Integrons are usually 2000), Chlamydia,(Dugan et al., 2004) and Salmonella part of larger transposons, which in turn are often part of (Carattoli et al., 2002) or in elements closely related to GIs conjugative plasmids or ICEs, to which we refer later. Despite in Haemophilus (Mohd-Zain et al., 2004). some transposons and integrons not being mobile, when Several intermediate elements can be found in bacteria. For they are found within a plasmid or an ICE, they can be dis- instance, conjugative plasmids can be the sites of the inser- persed by conjugation. tion of composite transposons, thereby allowing transposons Genomic islands (GIs) are more or less closely related to be transferred between cells (Beaber et al., 2002; Quintiliani to other MGEs and are found in several bacterial genera. and Courvalin, 1996). In fact, classification has become so 4 M. Brown-Jaque et al./Plasmid 79 (2015) 1–7 difficult that new terms have been proposed, such as Integra- et al., 2011; Modi et al., 2013; Quirós et al., 2014), human tive conjugative elements (ICEs). ICEs (Wozniak and Waldor, lung (Fancello et al., 2011) or waste water treatment plant 2010) refer to all the MGEs that can be mobilized through con- sludge (Calero-Cáceres et al., 2014; Parsley et al., 2010). jugation, without restricting this to plasmids, since it has become Because of their remarkable persistence, bacteriophages evident that conjugation systems are also widespread in appear to be suitable DNA vehicles in the more adverse ex- chromosome-borne MGEs. tracellular conditions (Muniesa et al., 2013a, 2013b). Their ICEs are similar to other MGEs, such as temperate phages survival capacity, together with the fact that polyvalent (which are integrated into and excised from the host chro- phages display a broad range of infectivity among differ- mosome but are not transmitted via conjugation), ent bacterial genera (Evans et al., 2010; Petrovski et al., 2011; transposons (which are integrated into and excised from Souza et al., 1972), makes bacteriophages especially suited the chromosome but are not transferred horizontally) and for movement between different biomes. plasmids (which are sometimes transferred from cell to cell Several examples of phages mobilizing ARGs can be found by conjugation but replicate autonomously). ICEs, unlike in the literature; P1-like phages have been reported to plasmids, cannot persist in an extrachromosomal state, as carry β-lactamase genes in Escherichia coli (Billard-Pomares they seem to be incapable of autonomous replication, al- et al., 2014) as have P22 phages in Salmonella (Schmieger though this still requires confirmation (Wozniak and Waldor, and Schicklmaier, 1999). Transduction of tetracycline re- 2010). sistance or acquisition of multiple resistance to chloram- ICEs can include conjugative transposons, which are in- phenicol, macrolide antibiotics, lincomycin and clindamycin tegrated DNA elements that excise themselves to form a or erythromycin occurred via phages in Streptococcus (Hyder covalently closed circular intermediate (Salyers et al., 1995), and Streitfeld, 1978; Ubukata et al., 1975). The tetracy- and represents an intermediate stage between plasmid and cline resistance gene has been transduced in Enterococcus transposon. In recent years, it has become apparent that the (Mazaheri Nezhad Fard et al., 2011). The prophage Wβ majority of tet(M) or cat genes among tetracycline- or encodes demonstrable fosfomycin resistance in Bacillus chloramphenicol-nonsusceptible pneumococci are con- anthracis (Schuch and Fischetti, 2006). tained within ICEs (Henderson-Begg et al., 2009), and Bacteriophages could mobilize genes by means of spe- examples can be found in Vibrio (Beaber et al., 2002; Waldor cialized or generalized transduction. Specialized transduction et al., 1996). involves the transfer of only a few specific genes, while generalized transduction mobilizes any fragment of the bacterial 2.2. MGEs that mediate ARG transfer without cell–cell genome (Bushman, 2002). Classically, it has been ac- contact cepted that those phages (λ, T7) that are capable of cutting and packaging their DNA by the “cos” mechanism, gener- Some ARG transfer mechanisms do not require direct cell- ating unit-length encapsidated DNA molecules, were to-cell contact since the MGE harboring the bacterial DNA responsible for specialized transduction. In contrast, those can be spread outside the donor cell and reach a suitable phages (P22, P1, T4) that package their DNA by cleavage at recipient. The persistence in the extracellular milieu of the a specific sequence in the phage genome (pac site) and fill element involved in the ARG transfer is then critical for its their capsids to capacity were able to mobilize bacterial DNA success. Some DNA fragments, including plasmids, can be by generalized transduction. In fact, generalized transduc- released from the donor cell and incorporated into the re- tion relies on the pac site homologues in the bacterial cipient by natural transformation, a mechanism that does chromosome that are “erroneously” recognized by the phage not require physical cell contact, but natural transforma- machinery and cleaved, allowing the package of bacterial tion does not seem to be widespread among bacterial species DNA in phage capsids. (Syvanen and Kado, 2001). Recent findings suggest, however, that the line that The most suitable vehicle for transfer between non- defines the types of transfer is not so clearly defined. The contiguous cells could be bacteriophages. In order to avoid genetic transfer of S. aureus GIs, currently mediated by pac restrictive classifications, this section will focus on phages phages, has recently been reported to involve either cos or and also on many intermediates that are, to some extent, pac phages (Quiles-Puchalt et al., 2014), thereby challeng- phage-related (Fig. 1). ing the classical paradigm of generalized transduction “Bacteria don’t die, just phage away”, the famous quote restricted only to pac phages. from the microbiologist Mark T Müller reflects the outcome Once the phage mobilizing ARGs by either mechanism of the process known as transduction. Bacterial genomes reaches the recipient bacteria, the survival of the bacterial are practically littered with DNA of viral origin, which ac- genes depends on the capability of the sequences to inte- counts for some 20% of the bacterial genome, including fully grate into the bacterial genome. If the ARG has been functional prophages or remnant phages. Recent reports in- mobilized in a specialized transducing phage, the com- dicate that phage implication in the transfer of ARGs could plete genomic content of the phage will include an integrase be more important than previously thought, particularly in gene, which will increase the chances of successful inte- the environment (American Academy of Microbiology, 2009). gration. If the particle is not a phage anymore, but a particle Recent studies show the presence of ARGs in phage par- derived from generalized transduction, then successful prev- ticles in the viral fraction of contaminated water bodies alence of the ARG will require recombination (either (Balcazar, 2014; Colomer-Lluch et al., 2011a, 2011b, 2014; homologous, illegitimate or site-specific recombination) Muniesa et al., 2004), as well as in complex environments into the host chromosome. The presence of genes encod- with high bacterial density, such as the human gut (Minot ing recombination and integration enzymes (e.g. Rec, M. Brown-Jaque et al./Plasmid 79 (2015) 1–7 5 recombinases or integrases from remnant prophages, 3.3. GI-phage transposases or topoisomerases, etc.) will be determinant for the frequency of acquisition of the ARGs (Brigulla and Bacteriophages of S. aureus that can efficiently package Wackernagel, 2010; Duesberg et al., 2008; Périchon and various bacterial genes and MGEs can also package the Courvalin, 2009). staphylococcal cassette chromosome mec (SCCmec) with dif- Some exceptions of this requirement of integration can ferent frequencies (Mašlanˇová et al., 2013). Thus, some of be found in the few phages (P1, N15, LE1, φ20 and φBB-1) the GIs described above as elements that require close that exist as circular or linear plasmids (Casjens, 2003). Of contact between cells to be mobilized could also be trans- these, the extra-chromosomal phages in S. aureus (ExPΦs) ferred without cell-to-cell contact by means of phages or have been reported to mobilize ARGs and also virulence phage-like elements. genes (Utter et al., 2014). 3.4. Gene transfer agents (GTA)

3. MGEs composed by phages or phage-related GTAs are tailed phage-like entities with the capacity to elements pack and transfer random pieces of the host genome (Guy et al., 2013; Lang et al., 2012). They mobilize bacterial DNA 3.1. Plasmid-phage in a process similar to that undergone by phage particles derived from generalized transduction (Bushman, 2002). In Pseudomonas aeruginosa transduction of imipenem, Nevertheless, a few differences should be pointed out. The aztreonam and ceftazidime is mediated by phages AP-1 and most remarkable is that no previous infection of the bac- AP-12. In addition, these genes could also be mobilized by terial host by a transducing phage is necessary to generate conjugation, suggesting a circular replicative form and in- GTAs. This is because the genes encoding GTA capsids are corporation in a conjugative plasmid (Blahová et al., 1993). already present in the bacterial chromosome, as a sort of It is not clear whether in this study phages have been found “usable” capsid element to mobilize bacterial DNA. The in a plasmid replicative form, an intermediate plasmidic genetic relationship between GTAs and phage genes is clear stage of the phage replication cycle or if they were actual- (Penadés et al., 2014), but some differences can also be ob- ly facing ICEs and generalized transduction events. Moreover, served. Mostly, GTAs have been reported in bacterial species plasmid pRYCE21 from Klebsiella pneumoniae strain KP4aC isolated from oceans and in general within alpha- contained a novel phage-related element immediately up- proteobacteria (Lang and Beatty, 2007; McDaniel et al., 2010, stream of blaCTX-M-10 (Oliver et al., 2005). This phage-related 2012). There are no reports of similar elements in other element is conserved among different blaCTX-M-10-producing groups; although it seems highly probable that there are strains of Enterobacteriaceae (Oliver et al., 2005). equivalents of these elements. The methodologies used for the determination of phage or phage-derived particles harboring ARGs in different 3.2. Transposon-phage studies that analyze the virome fraction of different samples (Balcazar, 2014; Colomer-Lluch et al., 2011b, 2014; Fancello Intermediate stages between transposons and et al., 2011; Modi et al., 2013) are based on the separation phage genomes can also be found. Actinobacillus of the different microbial fractions of the samples. The pro- actinomycetemcomitans contains a transposon that harbors cedures are based on isolating only phage particles and tetracyclin resistance and a temperate phage, named Aa phi avoiding the presence of non-encapsidated DNA (mostly bac- ST1 element (Willi et al., 1997). This phage can transduce terial) that could interfere with the results. It cannot be ruled tetracyclin resistance by mobilization of the Tn element out that in many of these studies some particles could be (Willi et al., 1997). Similarly, mefA, a gene encoding mac- derived from either specialized, generalized composite or rolide resistance in Streptococcus pyogenes, is located in a GTA elements, and these will be indistinguishable in the pool 58.8 kb genetic element, composed of a transposon. The that it is necessary to isolate to get the results. Consider- transposon is located within a prophage (Banks et al., 2003). ing that there are only a few reports of specialized phages Also in Streptococcus,Tn916-like, tet(M)-containing ele- harboring ARGs, it seems fair to assume that these should ments have been identiﬁed in plasmids or phages; but in not account for the most abundant fraction. Hence, either addition, evidence of antimicrobial resistance-conferring generalized particles or GTA particles, if present in some bac- Tn916-like elements has recently been reported within a terial groups such as Enterobacteriaceae or Bacteroidetes, pathogenicity island (Wyres et al., 2013). Moreover, phage although not yet reported as such, could both be detect- ϕC2 transduces erythromycin resistance between strains of able in this sort of analysis. Clostridium diﬃcile (Goh et al., 2013). The association between phages and other MGEs could Along different lines, transposable phages are those that be caused by the genetic relationships between them. This integrate within the chromosome, but can also move to dif- fact and their co-existence within the same bacterial cell ferent chromosomal sites as transposons do. A clear example could promote interactions and recombination events that can be found in phage Mu (Howe and Bade, 1975). The Tn21 generate the composite MGEs and phage-related ele- element which harbors different ARGs and is located in a ments. The reason why these elements once generated are plasmid has been found to share elements with the phage favorably selected, or if this selection is more remarkable Mu, among others (Rådström et al., 1994), which again sug- for those elements harboring ARGs than for other genes, gests that there is a close link between these MGEs. remains unknown. 6 M. Brown-Jaque et al./Plasmid 79 (2015) 1–7

4. Concluding remarks Brigulla, M., Wackernagel, W., 2010. Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues. Appl. Microbiol. Biotechnol. 86, 1027–1041. doi:10.1007/s00253-010- Unfortunately, the need for effective drugs has had a 2489-3. significant environmental downside. In the 60 years since Bushman, F., 2002. Lateral DNA Transfer: Mechanisms and Consequences. their introduction, millions of tons of antibiotics have been Cold Spring Harbor Laboratory Press, New York. Cabezón, E., Ripoll-Rozada, J., Peña, A., de la Cruz, F., Arechaga, I., 2014. produced and used for a wide variety of purposes. The planet Towards an integrated model of bacterial conjugation. FEMS Microbiol. is awash with these agents, which has clearly contributed Rev. doi:10.1111/1574-6976.12085. significantly to the selection of resistant strains and to the Calero-Cáceres, W., Melgarejo, A., Colomer-Lluch, M., Stoll, C., Lucena, F., Jofre, J., et al., 2014. Sludge as a potential important source of antibiotic movement of ARGs between biomes. To combat this spread, resistance genes in both the bacterial and bacteriophage fractions. several strategies are already starting to be implemented: Environ. Sci. Technol. 48, 7602–7611. doi:10.1021/es501851s. (1) the minimization of the use of antibiotics and the re- Cambray, G., Guerout, A.-M., Mazel, D., 2010. Integrons. Annu. Rev. Genet. 44, 141–166. doi:10.1146/annurev-genet-102209-163504. duction of their environmental expansion; (2) the financing Carattoli, A., Filetici, E., Villa, L., Dionisi, A.M., Ricci, A., Luzzi, I., 2002. of programs to synthetize new antimicrobial compounds; Antibiotic resistance genes and Salmonella genomic island 1 in (3) the exploration of new alternative therapies, such as Salmonella enterica serovar Typhimurium isolated in Italy. Antimicrob. the use of phages as antimicrobial agents; and finally Agents Chemother. 46, 2821–2828. Casjens, S., 2003. Prophages and bacterial genomics: what have we learned (4) the improvement of our knowledge of HGT strategies so far? Mol. Microbiol. 49, 277–300. with the aim of designing barriers to block the transfer of Chancey, S.T., Zähner, D., Stephens, D.S., 2012. Acquired inducible resistance. To succeed in this last point, we must face the antimicrobial resistance in Gram-positive bacteria. Future Microbiol. 7, 959–978. doi:10.2217/fmb.12.63. problem from a more varied perspective and with wider Colomer-Lluch, M., Imamovic, L., Jofre, J., Muniesa, M., 2011a. vision. Broad observation of the different gene transfer Bacteriophages carrying antibiotic resistance genes in fecal waste from mechanisms is necessary, because they share common fea- cattle, pigs, and poultry. Antimicrob. Agents Chemother. 55, 4908–4911. doi:10.1128/AAC.00535-11. tures and interact, but we also need to consider the Colomer-Lluch, M., Jofre, J., Muniesa, M., 2011b. Antibiotic resistance genes interconnection between different biomes (clinics, food and in the bacteriophage DNA fraction of environmental samples. PLoS ONE water, animals and the environment), as ARG transfer is 6, e17549. Colomer-Lluch, M., Jofre, J., Muniesa, M., 2014. Quinolone resistance genes not limited to one environment and not restricted to a single (qnrA and qnrS) in bacteriophage particles from wastewater samples field of research. and the effect of inducing agents on packaged antibiotic resistance genes. J. Antimicrob. Chemother. 69, 1265–1274. doi:10.1093/jac/ dkt528. Acknowledgments Darmon, E., Leach, D.R.F., 2014. Bacterial genome instability. Microbiol. Mol. Biol. Rev. 78, 1–39. doi:10.1128/MMBR.00035-13. This study was supported by the Spanish Ministry of Ed- Das, B., Martínez, E., Midonet, C., Barre, F.-X., 2013. Integrative mobile elements exploiting Xer recombination. Trends Microbiol. 21, 23–30. ucation and Science (AGL2012-30880), the Fundación Ramon doi:10.1016/j.tim.2012.10.003. Areces and the Generalitat de Catalunya (2009SGR1043). Davies, J., Davies, D., 2010. Origins and evolution of antibiotic resistance. WC-C has a PhD fellowship from SENESCYT-Republic of Microbiol. Mol. Biol. Rev. 74, 417–433. doi:10.1128/MMBR.00016-10. Davies, J.E., 1997. Origins, acquisition and dissemination of antibiotic Ecuador, MABJ has a grant from COLCIENCIAS (Republic of resistance determinants. Ciba Found. Symp. 207, 15–27, discussion Colombia). 27–35. Duesberg, C.B., Malhotra-Kumar, S., Goossens, H., McGee, L., Klugman, K.P., Welte, T., et al., 2008. Interspecies recombination occurs frequently in References quinolone resistance-determining regions of clinical isolates of Streptococcus pyogenes. Antimicrob. Agents Chemother. 52, 4191– American Academy of Microbiology, 2009. Antibiotic Resistance: An 4193. doi:10.1128/AAC.00518-08. Ecological Perspective on an Old Problem. Washington, DC. Dugan, J., Rockey, D.D., Jones, L., Andersen, A.A., 2004. Tetracycline resistance Balcazar, J.L., 2014. Bacteriophages as vehicles for antibiotic resistance genes in Chlamydia suis mediated by genomic islands inserted into the in the environment. PLoS Pathog. 10, e1004219. doi:10.1371/ chlamydial inv-like gene. Antimicrob. Agents Chemother. 48, 3989– journal.ppat.1004219. 3995. doi:10.1128/AAC.48.10.3989-3995.2004. Banks, D.J., Porcella, S.F., Barbian, K.D., Martin, J.M., Musser, J.M., 2003. Esposito, D., Scocca, J.J., 1997. The integrase family of tyrosine recombinases: Structure and distribution of an unusual chimeric genetic element evolution of a conserved active site domain. Nucleic Acids Res. 25, encoding macrolide resistance in phylogenetically diverse clones of 3605–3614. group A Streptococcus. J. Infect. Dis. 188, 1898–1908. doi:10.1086/ Evans, T.J., Crow, M.A., Williamson, N.R., Orme, W., Thomson, N.R., 379897. Komitopoulou, E., et al., 2010. Characterization of a broad-host-range Beaber, J.W., Hochhut, B., Waldor, M.K., 2002. Genomic and functional flagellum-dependent phage that mediates high-efficiency generalized analyses of SXT, an integrating antibiotic resistance gene transfer transduction in, and between, Serratia and Pantoea. Microbiology 156, element derived from Vibrio cholerae. J. Bacteriol. 184, 4259–4269. 240–247. doi:10.1099/mic.0.032797-0. Billard-Pomares, T., Fouteau, S., Jacquet, M.E., Roche, D., Barbe, V., Fancello, L., Desnues, C., Raoult, D., Rolain, J.M., 2011. Bacteriophages and Castellanos, M., et al., 2014. Characterization of a P1-like bacteriophage diffusion of genes encoding antimicrobial resistance in cystic fibrosis carrying an SHV-2 extended-spectrum β-lactamase from an Escherichia sputum microbiota. J. Antimicrob. Chemother. 66, 2448–2454. coli strain. Antimicrob. Agents Chemother. 58, 6550–6557. doi:10.1128/ doi:10.1093/jac/dkr315. AAC.03183-14. Gogarten, J.P., Townsend, J.P., 2005. Horizontal gene transfer, genome Blahová, J., Hupková, M., Babálová, M., Krcméry, V., Schäfer, V., 1993. innovation and evolution. Nat. Rev. Microbiol. 3, 679–687. doi:10.1038/ Transduction of resistance to Imipenem, Aztreonam and Ceftazidime nrmicro1204. in nosocomial strains of Pseudomonas aeruginosa by wild-type phages. Goh, S., Hussain, H., Chang, B.J., Emmett, W., Riley, T., V, Mullany, P., 2013. Acta Virol. 37, 429–436. Phage ϕC2 mediates transduction of Tn6215, encoding erythromycin Boccard, F., Smokvina, T., Pernodet, J.L., Friedmann, A., Guérineau, M., 1989. resistance, between Clostridium difficile strains. mBio 4, e00840-13. The integrated conjugative plasmid pSAM2 of Streptomyces doi:10.1128/mBio.00840-13. ambofaciens is related to temperate bacteriophages. EMBO J. 8, Guy, L., Nystedt, B., Toft, C., Zaremba-Niedzwiedzka, K., Berglund, E.C., 973–980. Granberg, F., et al., 2013. A gene transfer agent and a dynamic repertoire Boyd, E.F., Almagro-Moreno, S., Parent, M.A., 2009. Genomic islands are of secretion systems hold the keys to the explosive radiation of the dynamic, ancient integrative elements in bacterial evolution. Trends emerging pathogen Bartonella. PLoS Genet. 9, e1003393. doi:10.1371/ Microbiol. 17, 47–53. doi:10.1016/j.tim.2008.11.003. journal.pgen.1003393. M. Brown-Jaque et al./Plasmid 79 (2015) 1–7 7

Henderson-Begg, S.K., Roberts, A.P., Hall, L.M.C., 2009. Diversity of putative Parsley, L.C., Consuegra, E.J., Kakirde, K.S., Land, A.M., Harper, W.F., Liles, Tn5253-like elements in Streptococcus pneumoniae. Int. J. Antimicrob. M.R., 2010. Identification of diverse antimicrobial resistance Agents 33, 364–367. doi:10.1016/j.ijantimicag.2008.10.002. determinants carried on bacterial, plasmid, or viral metagenomes from Howe, M.M., Bade, E.G., 1975. Molecular biology of bacteriophage mu. an activated sludge microbial assemblage. Appl. Environ. Microbiol. 76, Science 190, 624–632. 3753–3757. doi:10.1128/AEM.03080-09. Hyder, S.L., Streitfeld, M.M., 1978. Transfer of erythromycin resistance from Périchon, B., Courvalin, P., 2009. VanA-type vancomycin-resistant clinically isolated lysogenic strains of Streptococcus pyogenes via their Staphylococcus aureus. Antimicrob. Agents Chemother. 53, 4580–4587. endogenous phage. J. Infect. Dis. 138, 281–286. doi:10.1128/AAC.00346-09. Juhas, M., van der Meer, J.R., Gaillard, M., Harding, R.M., Hood, D.W., Crook, Penadés, J.R., Chen, J., Quiles-Puchalt, N., Carpena, N., Novick, R.P., 2014. D.W., 2009. Genomic islands: tools of bacterial horizontal gene transfer Bacteriophage-mediated spread of bacterial virulence genes. Curr. Opin. and evolution. FEMS Microbiol. Rev. 33, 376–393. doi:10.1111/j.1574- Microbiol. 23C, 171–178. doi:10.1016/j.mib.2014.11.019. 6976.2008.00136.x. Petrovski, S., Seviour, R.J., Tillett, D., 2011. Characterization of the genome Katayama, Y., Ito, T., Hiramatsu, K., 2000. A new class of genetic element, of the polyvalent lytic bacteriophage GTE2, which has potential for staphylococcus cassette chromosome mec, encodes methicillin biocontrol of Gordonia-, Rhodococcus-, and Nocardia-stabilized foams resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. in activated sludge plants. Appl. Environ. Microbiol. 77, 3923–3929. 44, 1549–1555. doi:10.1128/AEM.00025-11. Keeling, P.J., Palmer, J.D., 2008. Horizontal gene transfer in eukaryotic Quiles-Puchalt, N., Carpena, N., Alonso, J.C., Novick, R.P., Marina, A., Penadés, evolution. Nat. Rev. Genet. 9, 605–618. doi:10.1038/nrg2386. J.R., 2014. Staphylococcal pathogenicity island DNA packaging system Lang, A.S., Beatty, J.T., 2007. Importance of widespread gene transfer agent involving cos-site packaging and phage-encoded HNH endonucleases. genes in alpha-proteobacteria. Trends Microbiol. 15, 54–62. Proc. Natl. Acad. Sci. U.S.A. 111, 6016–6021. doi:10.1073/ doi:10.1016/j.tim.2006.12.001. pnas.1320538111. Lang, A.S., Zhaxybayeva, O., Beatty, J.T., 2012. Gene transfer agents: Quintiliani, R., Courvalin, P., 1996. Characterization of Tn1547, a composite phage-like elements of genetic exchange. Nat. Rev. Microbiol. 10, transposon flanked by the IS16 and IS256-like elements, that confers 472–482. doi:10.1038/nrmicro2802. vancomycin resistance in Enterococcus faecalis BM4281. Gene 172, 1–8. Mašlanˇová, I., Doškarˇ, J., Varga, M., Kuntová, L., Mužík, J., Malúšková, D., Quirós, P., Colomer-Lluch, M., Martínez-Castillo, A., Miró, E., Argente, M., et al., 2013. Bacteriophages of Staphylococcus aureus efficiently package Jofre, J., et al., 2014. Antibiotic resistance genes in the bacteriophage various bacterial genes and mobile genetic elements including SCCmec DNA fraction of human fecal samples. Antimicrob. Agents Chemother. with different frequencies. Environ. Microbiol. Rep. 5, 66–73. 58, 606–609. doi:10.1128/AAC.01684-13. doi:10.1111/j.1758-2229.2012.00378.x. Rådström, P., Sköld, O., Swedberg, G., Flensburg, J., Roy, P.H., Sundström, Mazaheri Nezhad Fard, R., Barton, M.D., Heuzenroeder, M.W., 2011. L., 1994. Transposon Tn5090 of plasmid R751, which carries an Bacteriophage-mediated transduction of antibiotic resistance in integron, is related to Tn7, Mu, and the retroelements. J. Bacteriol. 176, enterococci. Lett. Appl. Microbiol. 52, 559–564. doi:10.1111/j.1472- 3257–3268. 765X.2011.03043.x. Salyers, A.A., Shoemaker, N.B., Stevens, A.M., Li, L.Y., 1995. Conjugative McDaniel, L.D., Young, E., Delaney, J., Ruhnau, F., Ritchie, K.B., Paul, J.H., 2010. transposons: an unusual and diverse set of integrated gene transfer High frequency of horizontal gene transfer in the oceans. Science 330, elements. Microbiol. Rev. 59, 579–590. 50. doi:10.1126/science.1192243. Schmieger, H., Schicklmaier, P., 1999. Transduction of multiple drug McDaniel, L.D., Young, E.C., Ritchie, K.B., Paul, J.H., 2012. Environmental resistance of Salmonella enterica serovar typhimurium DT104. FEMS factors influencing gene transfer agent (GTA) mediated transduction Microbiol. Lett. 170, 251–256. in the subtropical ocean. PLoS ONE 7, e43506. doi:10.1371/ Schuch, R., Fischetti, V.A., 2006. Detailed genomic analysis of the Wbeta journal.pone.0043506. and gamma phages infecting Bacillus anthracis: implications for Minot, S., Sinha, R., Chen, J., Li, H., Keilbaugh, S.A., Wu, G.D., et al., 2011. evolution of environmental fitness and antibiotic resistance. J. Bacteriol. The human gut virome: inter-individual variation and dynamic 188, 3037–3051. doi:10.1128/JB.188.8.3037-3051.2006. response to diet. Genome Res. 21, 1616–1625. doi:10.1101/ Souza, K.A., Ginoza, H.S., Haight, R.D., 1972. Isolation of a polyvalent gr.122705.111. bacteriophage for Escherichia coli, Klebsiella pneumoniae, and Modi, S.R., Lee, H.H., Spina, C.S., Collins, J.J., 2013. Antibiotic treatment Aerobacter aerogenes. J. Virol. 9, 851–856. expands the resistance reservoir and ecological network of the phage Syvanen, M., Kado, C.I., 2001. Horizontal Gene Transfer. Academic Press. metagenome. Nature 499, 219–222. doi:10.1038/nature12212. Ubukata, K., Konno, M., Fujii, R., 1975. Transduction of drug resistance to Mohd-Zain, Z., Turner, S.L., Cerdeño-Tárraga, A.M., Lilley, A.K., Inzana, T.J., tetracycline, chloramphenicol, macrolides, lincomycin and clindamycin Duncan, A.J., et al., 2004. Transferable antibiotic resistance elements with phages induced from Streptococcus pyogenes. J. Antibiot. (Tokyo) in Haemophilus influenzae share a common evolutionary origin with 28, 681–688. a diverse family of syntenic genomic islands. J. Bacteriol. 186, 8114– Utter, B., Deutsch, D.R., Schuch, R., Winer, B.Y., Verratti, K., Bishop-Lilly, 8122. doi:10.1128/JB.186.23.8114-8122.2004. K., et al., 2014. Beyond the chromosome: the prevalence of unique Muniesa, M., García, A., Miró, E., Mirelis, B., Prats, G., Jofre, J., et al., 2004. extra-chromosomal bacteriophages with integrated virulence genes Bacteriophages and diffusion of beta-lactamase genes. Emerg. Infect. in pathogenic Staphylococcus aureus. PLoS ONE 9, e100502. Dis. 10, 1134–1137. doi:10.3201/eid1006.030472. doi:10.1371/journal.pone.0100502. Muniesa, M., Schembri, M.A., Hauf, N., Chakraborty, T., 2006. Active genetic Waldor, M.K., Tschäpe, H., Mekalanos, J.J., 1996. A new type of conjugative elements present in the locus of enterocyte effacement in Escherichia transposon encodes resistance to sulfamethoxazole, trimethoprim, coli O26 and their role in mobility. Infect. Immun. 74, 4190–4199. and streptomycin in Vibrio cholerae O139. J. Bacteriol. 178, 4157– doi:10.1128/IAI.00926-05. 4165. Muniesa, M., Colomer-Lluch, M., Jofre, J., 2013a. Potential impact of Willi, K., Sandmeier, H., Kulik, E.M., Meyer, J., 1997. Transduction of environmental bacteriophages in spreading antibiotic resistance genes. antibiotic resistance markers among Actinobacillus actinomy- Future Microbiol. 8, 739–751. doi:10.2217/fmb.13.32. cetemcomitans strains by temperate bacteriophages Aa phi 23. Cell. Muniesa, M., Colomer-Lluch, M., Jofre, J., 2013b. Could bacteriophages Mol. Life Sci. 53, 904–910. transfer antibiotic resistance genes from environmental bacteria to Wozniak, R.A.F., Waldor, M.K., 2010. Integrative and conjugative elements: human-body associated bacterial populations? Mob. Genet. Elements mosaic mobile genetic elements enabling dynamic lateral gene flow. 3, e25847. doi:10.4161/mge.25847. Nat. Rev. Microbiol. 8, 552–563. doi:10.1038/nrmicro2382. Oliver, A., Coque, T.M., Alonso, D., Valverde, A., Baquero, F., Cantón, R., 2005. Wyres, K.L., van Tonder, A., Lambertsen, L.M., Hakenbeck, R., Parkhill, J., CTX-M-10 linked to a phage-related element is widely disseminated Bentley, S.D., et al., 2013. Evidence of antimicrobial resistance- among Enterobacteriaceae in a Spanish hospital. Antimicrob. Agents conferring genetic elements among pneumococci isolated prior to 1974. Chemother. 49, 1567–1571. doi:10.1128/AAC.49.4.1567-1571.2005. BMC Genomics 14, 500. doi:10.1186/1471-2164-14-500.