How do bacteriophages promote antibiotic resistance in the environment?

Antibiotic resistance has become a major global health concern because the increasing prevalence of this phenomenon is compromising the effectiveness of antimicrobial therapy. Around 700 000 deaths worldwide are attributed annually to antibiotic-resistant infections (http://amr-review.org). Bacteria can acquire antibiotic resistance through chromosomal mutations or the acquisition of genetic material from other bacteria or the environment via horizontal gene transfer. This latter process is largely driven by mobile genetic elements, such as plasmids, transposons or bacteriophages, which play an essential role in the evolution and ecology of bacterial communities by controlling the intraspecies and interspecies exchange of genetic information [[1]Frost L.S. Leplae R. Summers A.O. Toussaint A. Mobile genetic elements: the agents of open source evolution.Nat Rev Microbiol. 2005; 3: 722-732Crossref PubMed Scopus (1035) Google Scholar]. While the transfer of these mobile genetic elements may occur through transformation or transduction, conjugation is considered the most efficient mechanism to exchange genetic material among bacteria [[2]Courvalin P. Transfer of antibiotic resistance genes between Gram-positive and Gram-negative bacteria.Antimicrob Agents Chemother. 1994; 38: 1447-1451Crossref PubMed Scopus (250) Google Scholar]. Because antibiotic resistance genes (ARGs) are acquired and frequently spread by conjugation through conjugative plasmids and transposons, the contribution of these elements to antibiotic resistance has been extensively studied in hospital and community settings [3Hardiman C.A. Weingarten R.A. Conlan S. Khil P. Dekker J.P. Mathers A.J. et al.Horizontal transfer of carbapenemase-encoding plasmids and comparison with hospital epidemiology data.Antimicrob Agents Chemother. 2016; 60: 4910-4919Crossref PubMed Scopus (49) Google Scholar, 4Chen L. Chavda K.D. Melano R.G. Hong T. Rojtman A.D. Jacobs M.R. et al.Molecular survey of the dissemination of two blaKPC-harboring IncFIA plasmids in New Jersey and New York hospitals.Antimicrob Agents Chemother. 2014; 58: 2289-2294Crossref PubMed Scopus (53) Google Scholar]. However, little is known about the role of bacteriophages as vehicles for ARGs in environmental settings. Recent findings based on cutting-edge genomic technologies suggest that, in these settings, bacteriophages play a more important role in the mobilization of ARGs than previously expected. Bacteriophages (phages) are viruses that infect bacteria and have the ability to transfer genetic material between bacteria via transduction. Broadly speaking, they may be grouped according to their life cycle: lytic phages and lysogenic (temperate) phages. After lytic phages infect their bacterial host, the phage genome is replicated and assembled into phage particles that are released through cell lysis. Temperate phages integrate their genetic material into the bacterial chromosome as prophages, persisting in a latent or dormant state without causing cell lysis [[5]Feiner R. Argov T. Rabinovich L. Sigal N. Borovok I. Herskovits A.A. A new perspective on lysogeny: prophages as active regulatory switches of bacteria.Nat Rev Microbiol. 2015; 13: 641-650Crossref PubMed Scopus (251) Google Scholar]. Phages have the potential to transfer genetic material between hosts using either generalized or specialized transduction (Fig. 1). Generalized transduction is the process by which bacterial DNA fragments are randomly packaged into the capsid during the lytic cycle, whereas specialized transduction is limited to temperate phages that integrate their genomes into the host chromosome at specific locations. Some temperate phages may encode factors that increase the fitness and survival of their hosts in a process known as lysogenic conversion. As a consequence, phages have emerged as prime suspects in bacterial adaptation and evolution by facilitating the exchange of genetic material. These characteristics make them suitable vehicles for acquisition, maintenance and spread of antibiotic resistance determinants. A recent study demonstrated a relatively high prevalence of integron–integrase genes (i.e. intI1, intI2 and intI3) and genes conferring resistance to tetracyclines and β-lactam antibiotics (i.e. tetA, tetW, blaOXA and blaTEM genes) in phages isolated from different environmental sources [[6]Anand T. Bera B.C. Vaid R.K. Barua S. Riyesh T. Virmani N. et al.Abundance of antibiotic resistance genes in environmental bacteriophages.J Gen Virol. 2016; 97: 3458-3466Crossref PubMed Scopus (33) Google Scholar]. A high prevalence of the blaCTX-M-15 gene, which encodes resistance to extended-spectrum β-lactam antibiotics, was also detected in Escherichia coli phages from sewage samples [[7]Roshini J. Raj M. Karunasagar I. Prevalence of blaCTX-M-15 in coliphages isolated from sewage.Adv Sci Lett. 2017; 23: 1869-1871Crossref Scopus (3) Google Scholar]. Interestingly, a comparative study revealed that the abundance of genes conferring resistance to β-lactams (blaTEM, blaNDM and blaKPC) and glycopeptides (vanA) was increased in phage and plasmid DNA but not in the bacterial DNA fraction from samples collected downstream of wastewater treatment plant discharges [[8]Lekunberri I. Villagrasa M. Balcázar J.L. Borrego C.M. Contribution of bacteriophage and plasmid DNA to the mobilization of antibiotic resistance genes in a river receiving treated wastewater discharges.Sci Total Environ. 2017; 601–602: 206-209Crossref PubMed Scopus (71) Google Scholar]. Although previous studies have shown that human-associated viromes rarely carry genes conferring resistance to antibiotics [[9]Enault F. Briet A. Bouteille L. Roux S. Sullivan M.B. Petit M.A. Phages rarely encode antibiotic resistance genes: a cautionary tale for virome analyses.ISME J. 2017; 11: 237-247Crossref PubMed Scopus (195) Google Scholar], a recent study revealed that viromes from both anthropogenically impacted and non-impacted aquatic environments contain a large reservoir of resistance genes, including those conferring multidrug resistance to at least three different antibiotics [[10]Lekunberri I. Subirats J. Borrego C.M. Balcázar J.L. Exploring the contribution of bacteriophages to antibiotic resistance.Environ Pollut. 2017; 220: 981-984Crossref PubMed Scopus (88) Google Scholar]. Similar results have been observed in aquatic viromes from the Lambro River (Italy), whose relative abundance of reads associated with ARGs ranged from 0.48% to 1.92% [[11]Colombo S. Arioli S. Neri E. Della Scala G. Gargari G. Mora D. Viromes as genetic reservoir for the microbial communities in aquatic environments: a focus on antimicrobial-resistance genes.Front Microbiol. 2017; 81095Crossref PubMed Scopus (26) Google Scholar]. Likewise, an extensive study of viromes from different aquarium systems revealed the presence of genes conferring resistance to several antibiotic classes, with trimethoprim being the most common [[12]Kim Y. Van Bonn W. Aw T.G. Rose J.B. Aquarium viromes: viromes of human-managed aquatic systems.Front Microbiol. 2017; 81231Crossref PubMed Scopus (22) Google Scholar]. Considering that environmental settings are frequently exposed to antibiotic residues and resistant organisms from anthropogenic sources, such impacts may trigger ecological interactions between phages and their hosts. In fact, phages can provide their bacterial hosts with a substantial advantage under challenging conditions. A comparative study showed that the virome of antibiotic-treated mice was highly enriched for genes conferring resistance not only to the administered antibiotic but also to other antibiotics [[13]Modi S.R. Lee H.H. Spina C.S. Collins J.J. Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome.Nature. 2013; 499: 219-222Crossref PubMed Scopus (358) Google Scholar]. Although transduction is a mechanism of horizontal gene transfer associated with phages, a recent study has demonstrated that certain phages (lytic phages) may also be able to promote transformation [[14]Keen E.C. Bliskovsky V.V. Malagon F. Baker J.D. Prince J.S. Klaus J.S. et al.Novel "superspreader" bacteriophages promote horizontal gene transfer by transformation.mBio. 2017; 8: e02115-e02116Crossref PubMed Scopus (79) Google Scholar], whose process involves the uptake of naked DNA and recombination. Homologous recombination and DNA-repair processes normally limit the feasibility and success of transformation to DNA from closely related bacteria; however, ARGs may be potentially spread via broad-host-range plasmids without the need for recombination [[15]Thomas C.M. Nielsen K.M. Mechanisms of, and barriers to, horizontal gene transfer between bacteria.Nat Rev Microbiol. 2005; 3: 711-721Crossref PubMed Scopus (1283) Google Scholar]. It should be noted that many bacteria carry plasmids, extrachromosomal genetic elements that frequently encode antibiotic resistance, which can be released during phage infection and can subsequently be acquired by other bacteria through transformation. Altogether, these studies demonstrate that phages might play an important role in the acquisition and spread of ARGs into the environment. Several studies have also demonstrated that municipal wastewater treatment plants are hot spots for antibiotic-resistant bacteria and their ARGs, which are eventually released into receiving water bodies [16Rizzo L. Manaia C. Merlin C. Schwartz T. Dagot C. Ploy M.C. et al.Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review.Sci Total Environ. 2013; 447: 345-360Crossref PubMed Scopus (1533) Google Scholar, 17Zhu Y.G. Gillings M. Simonet P. Stekel D. Banwart S. Penuelas J. Microbial mass movements.Science. 2017; 357: 1099-1100Crossref PubMed Scopus (192) Google Scholar]; therefore, this problem can be amplified because phages can survive wastewater treatment processes better than bacteria [18Mocé-Llivina L. Muniesa M. Pimenta-Vale H. Lucena F. Jofre J. Survival of bacterial indicator species and bacteriophages after thermal treatment of sludge and sewage.Appl Environ Microbiol. 2003; 2003: 1452-1456Crossref Scopus (122) Google Scholar, 19Calero-Cáceres W. Muniesa M. Persistence of naturally occurring antibiotic resistance genes in the bacteria and bacteriophage fractions of wastewater.Water Res. 2016; 95: 11-18Crossref PubMed Scopus (104) Google Scholar]. Given this, further studies are required to elucidate the extent to which phages contribute to the mobilization of ARGs in environmental settings. A better understanding of the factors and mechanisms that promote environmental antibiotic resistance dissemination via phages will allow us to reach these goals. No conflicts of interest have been declared. This work was supported by the Generalitat de Catalunya (2014 SGR 291) and the European Union through the European Regional Development Fund (FEDER).