The antibiotic resistance genes that accumulate in the genomes of bacteria are likely to have their origins in disparate reservoirs, a conclusion arrived at in several independent recent studies on the evolution of MDR (Aminov, 2011; Amos et al., 2014; Guerra et al., 2014; Hsu et al., 2014; Ojer-Usoz et al., 2014) and evidenced also by the widespread dissemination of various antibiotic resistance genes, including those encoding resistance to extended spectrum β-lactamases in birds and other wildlife species (Costa et al., 2008; Poeta et al., 2008; Simoes et al., 2010). Aquatic and soil environments serve as sinks of older generation antibiotics including sulphonamides, tetracycline, amoxicillin, ampicillin and trimethoprim (Boxall, 2004; Monteiro and Boxall, 2009). The pools of antibiotic resistance genes available for dissemination (known as the “resistome”) are enriched by veterinary and agricultural practices, human sewage and hospital waste (Tennstedt et al., 2005; Galvin et al., 2010; Heuer et al., 2011; Wellington et al., 2013; Hsu et al., 2014; Ojer-Usoz et al., 2014).

Genes imparting antibiotic resistance move within bacterial population(s) through LGT, independent of the linear “parent to progeny” descent (Tennstedt et al., 2005; Stokes and Gillings, 2011). The genes can be from a wide “gene pool” and can be transmitted between closely and distantly related species. Examples in which LGT plays a significant role in the evolution of important multi-resistant pathogenic bacterial genomes include, but are not restricted to, E. coli, Pseudomonas aeruginosa, and Vibrio cholerae. Notably, these organisms can survive in a range of environmental niches outside of the human body, including fomites in hospitals, soil and aquatic environments. Consequently, there exists diverse niche-adapted pools of microbial communities that can provide opportunities for the sampling and exchange of genetic information. Further, these opportunities are likely to be influenced significantly by the ecology of the niche-adapted microbial communities (Heuer et al., 2009, 2011; Galvin et al., 2010; Forsberg et al., 2012; Wellington et al., 2013).

Reservoirs of antibiotic resistance genes that are increasingly recognized as important are the gastrointestinal tract of food animals, soil and water. This is because of the practice of using sub-therapeutic doses of antibiotics to promote the growth of food animals and the prophylactic use of antibiotics to prevent disease in food animals. A significant percentage of administered antibiotics are excreted in animal waste. Un-metabolized antibiotics concentrate in animal waste ponds and, depending on their chemical composition, partition to different fractions during the wastewater treatment processes (Kolpin et al., 2002; Ghosh and Lapara, 2007; Watanabe et al., 2010; Cheng et al., 2013; Li et al., 2013). Thus, antibiotics enter into environments/ecosystems where bacteria pathogenic to humans may also survive (Sarmah et al., 2006). The antibiotics used with food animals for growth promotion and prophylactically include a set of FDA (FDA, 2011) approved drugs, recognized by the World Health Organization as important to treat human diseases (Table 1) (Aarestrup et al., 2008). Further, the quantities of antibiotics administered to food animals are very large. In Australia, in the period 2005 to 2010, 98% of veterinary antibiotics sold were for use in food animals. Only approximately 43% were sold for therapeutic or prophylactic purposes (no distinction could be drawn between “therapeutic” and “prophylactic” uses as it was difficult for those surveyed to estimate the proportions of products used for these purposes). This means that, on average, for the surveyed period, sales of antimicrobials for growth promotion averaged 35.3 tons; in 2005–2006 this was as much as 47.2 tons (APVMA, 2014). Importantly, a large proportion of the antimicrobials sold (almost 77%), were administered in feed.

The practice of using sub-therapeutic doses of antibiotics to promote growth of food animals became widespread following initial studies of the effect of antibiotics on the growth of broiler chickens (Elam et al., 1953; Jacobs et al., 1953; Izat et al., 1990; Dibner and Richards, 2005; Castanon, 2007). Antibiotics are used for growth promotion in doses lower than the recommended minimum effective concentrations (MEC) for therapeutic purposes but often at concentrations greater than the minimum inhibitory concentration (MIC) of specific drug/microbe combinations (Berrang et al., 2007; Alexander et al., 2008; Mirzaagha et al., 2011; Holman and Chenier, 2013). The practice of using antibiotics to promote growth of food animals has been banned in several countries in Europe due to concerns over the development and spread of antimicrobial resistance (Gilbert, 2011; Maron et al., 2013).

The practice raises concerns over the development and spread of antimicrobial resistance because sub-inhibitory concentrations of antibiotics can enhance LGT (Barr et al., 1986; Torres et al., 1991; Stevens et al., 1993); potentially act as signal transduction molecules (Romero et al., 2011) in the transition from planktonic to biofilm phase during the process of monomicrobial and polymicrobial biofilm formations,(Bagge et al., 2004; Hoffman et al., 2005); and globally modulate transcriptional activity. Sub-therapeutic doses of β-lactam antibiotics has been shown to enhance the rate of conjugative plasmid transfer (Barr et al., 1986) and tetracycline has been implicated in driving the lateral transfer of integrative conjugative elements (Torres et al., 1991; Stevens et al., 1993). Conjugative transfer of single-stranded (ss) DNA (the mechanism by which plasmids are transferred from donor to recipient cells) induces the bacterial SOS response, which in turn up-regulates expression of the integron integrase resulting in the capture or exchange of resistance genes in Gram-negative pathogens (Baharoglu et al., 2010). Integron-mediated gene shuffling events in V. cholerae and E. coli were elevated significantly (4.5-fold and 37-fold increase respectively) in response to antibiotic mediated stress, which in turn triggered the SOS response. In addition, the study also reported up-regulation of the frequency of integron-associated excision/shuffling of gene-cassettes by 141-fold in V. cholera and 340-fold in E. coli, in a SOS response mediated event. In a recent study, Zhu et al. (2013) demonstrated the presence and relative abundance (an increase of 198-fold on average) of 149 antibiotic resistance genes (of 244 included in the study) conferring resistance to common in-feed and veterinary antibiotics used in the swine farming industry in China within 36 metagenomic samples extracted from compost, manure and soil. The study also provided evidence of abundance and enrichment of common transposase genes [identified in the course of a previous study (Aziz et al., 2010)] known to be frequently associated with antibiotic resistance genes. The conclusions of the study included a statement of the likely roles played by the subset of mobile elements in LGT of resistance genes in China (Zhu et al., 2013).

In a pioneering transcriptome analyses study, Goh et al. provided direct evidence of the global modulation of transcriptional activity of genes in Salmonella enterica serovar Typhimurium in the presence of sub-inhibitory concentrations of antibiotics like erythromycin (used as Antimicrobial growth promoters or AGP for poultry and swine) and rifampicin (Goh et al., 2002). Further gene-expression studies have now presented data on the influence of sub-inhibitory concentrations of antibiotics on the global transcriptome of genes related to virulence (Subrt et al., 2011), colonization (Bagge et al., 2004), motility, SOS stress response (inducible DNA repair system) and biofilm formation (Kaplan et al., 2012) for many important human pathogens (Davies et al., 2006). Notably, stress responses have been shown to enhance bacterial adaptation through increased mutation and LGT (Aertsen and Michiels, 2006; Baharoglu and Mazel, 2011).

Genes that encode resistance to antibiotics often appear clustered in the genomes of many bacteria, forming CRL (Parkhill et al., 2001; Szczepanowski et al., 2005, 2009; Tennstedt et al., 2005; Roy Chowdhury et al., 2009, 2011; Venturini et al., 2010; Toleman and Walsh, 2011). In addition to antibiotic resistance genes, the CRL possess diverse mobile genetic elements (predominantly insertion elements, transposons and integrons) clustered either in independently replicating units, like plasmids, or in genomic islands within the bacterial chromosome. Mobile elements facilitate dissemination of CRL within bacteria. Acquisition of a CRL by any bacterium thus provides a survival mechanism to it when exposed to an entire range of antimicrobial agents. CRL evolve rapidly, especially within Gram-negative bacteria, and they generally contain hotspots where additional resistance genes can accumulate. Lateral transfer of genes within the gastrointestinal tracts of humans and food animals, and in reservoirs where environmental bacteria mix with bacteria derived from anthropogenic activities (sewage, hospital waste and food production animal waste) significantly contribute to these evolutionary processes (Forsberg et al., 2012). Although mobile genetic elements are a key component of CRL, there is currently limited understanding of the different types of mobile genetic element(s) that may contribute to: (1) the spread of CRL; (2) the diversity of resistance gene reservoirs that can be utilized by the mobile elements to form CRL and (3) the mechanisms by which resistance genes assemble on laterally mobile segments of DNA.

Integrons represent a group of genetic elements that are most commonly found in association with CRL and have been implicated in the rapid evolution of multi-drug resistance within Gram-negative pathogens (Martinez et al., 2013). Based on the amino acid sequences of the integrase/recombinase gene intI (Recchia and Hall, 1995) integrons can be differentiated into several classes, all of which are found in diverse microbial communities (Marquez et al., 2008). Classes 1–3 are most frequently associated with the dissemination of MDR within Gram-negative bacteria, although recently class 1 integrons have also been identified in Gram-positive bacteria (Nandi et al., 2004; Shi et al., 2006; Xu et al., 2010, 2011). Exchange of resistance genes within different classes of integrons (from diverse bacteria) has been reported in a range of microbial communities (Labbate et al., 2008; Roy Chowdhury et al., 2009). The role of class 1 integrons in the evolution of MDR within hospitals is most comprehensively documented (Djordjevic et al., 2013; Martinez et al., 2013), although they are also frequently reported from environmental bacteria (Holmes et al., 2003; Stokes et al., 2006). Functionally, a class 1 integron is a two-component site-specific gene recombination system. Structurally a class 1 integron comprises an integrase gene and a tandem array of a variable number of independently acquired mobile genes. The independently mobile units of a class 1 integron are called “gene cassettes” and are made up of a promoter-less open reading frame and a recombination site. Class 1 integrons most frequently contain multiple gene cassettes, in structures called “cassette arrays,” that can be expressed from a single promoter (Stokes and Hall, 1989) in an operon-like manner. Exchange/insertion/deletion of gene cassettes (by the process of site-specific recombination) and expression of these genes enables their host to exhibit multiple “acquired” resistance phenotypes simultaneously and relatively quickly (on an evolutionary time scale).

A key component of the integron integrase is the integrase promoter (P_int) (Stokes and Hall, 1989). The regulation and expression of the integron integrase is an adaptive response to stress, including that induced by sub-lethal dosages of antibiotics in any environment (Guerin et al., 2009). Hocquet et al. described a metronidazole induced cassette rearrangement in a class 1 integron that resulted in the formation of a fused gcuF1-bla_OXA−28 cassette, which subsequently led to the expression of the fused β-lactamase gene and ceftazidime resistance in a P. aeruginosa isolate (Hocquet et al., 2012). A LexA binding site is found conserved across the vast majority of integrase genes, indicating the likelihood of bacterial SOS mediated up-regulation of most integron integrases (Guerin et al., 2009). Importance of the LexA binding site has been experimentally demonstrated in Vibrio spp. and E. coli (Guerin et al., 2009).

Class 1 integrons isolated from clinical samples, are predominantly linked to the Tn3 family of transposons, particularly Tn21 (Liebert et al., 1999) and Tn1696 (Partridge et al., 2001), which in turn harbor clustered mercury resistance genes. Class 1 integrons also have a propensity of targeting resolution sites of plasmids or other transposons on plasmid backbones (Kholodii et al., 1995; Minakhina et al., 1999). Thus, in addition to carrying genes conferring MDR, class 1 integrons also associate with other mobile elements, which independently carry other combinations of resistance genes to form laterally mobile resistance scaffolds (Parkhill et al., 2001; Szczepanowski et al., 2005; Tennstedt et al., 2005; Wibberg et al., 2013).

Plasmids play a central role in the lateral transfer of CRL within both closely and distantly related Gram-negative bacteria. Identical CRL are frequently seen piggy-backing on different plasmid backbones isolated from different hosts, supporting the role of plasmids in the transfer of drug resistance loci. Such events are best exemplified by plasmids carrying identical complex Tn21-associated MDR regions (Tennstedt et al., 2005; Szczepanowski et al., 2009; Venturini et al., 2013). Recent examples of such plasmids with complex resistance loci isolated from waste water treatment plants include IncF plasmids pRSB225 (Wibberg et al., 2013) and pRSB107 (Szczepanowski et al., 2005) and the series of IncP-1 plasmids reviewed recently by Popowska and Krawczyk-Balska (2013). Although isolated from waste water treatment plants, all these plasmids and a set of 140 plasmids described in a recent plasmid metagenomic study by Szczepanowski et al. clearly indicate that the resistance gene pool within the CRL on the plasmids are an admixture of genes that have originated from different bacterial communities (Szczepanowski et al., 2009).

The co-selection of antibiotic resistance genes by plasmids containing transposons with genes encoding resistance to heavy metals (such as mercury, cadmium, copper, and zinc) adds to the complexity of the evolution of laterally mobile clustered resistance regions (Seiler and Berendonk, 2012). This is particularly so when considering food animals and their environments (e.g. soil and water) as reservoirs of antibiotic resistance determinants; metals like copper and zinc, all of which are often used in animal husbandry for growth promotion (Jacob et al., 2010). This phenomenon of co-selection of resistance genes on plasmids increase the opportunity for microbial populations, that proliferate both in the gastrointestinal tracts of mammals and that survive in soil and aquatic environments, to act as major conduits in the flow of resistance genes between these reservoirs.

Plasmids pO26-CRL, pO26-CRL₁₂₅ (isolated from an enterohemorrhagic E. coli specimen from a human with bloody diarrhea) and pO111-CRL₁₁₅ (isolated from a bovine source in Australia) are examples of plasmids with identical clustered antibiotic and metal resistance genes that have been identified recently by our group (Venturini et al., 2010, 2013). The CRL in these plasmids are characterized by a Tn21-associated class 1 integron truncated by the presence of a composite IS26 transposon, Tn6026 (Cain et al., 2010). Composite transposons, another type of mobile element frequently associated with CRL, are defined as segments of DNA bounded by two related insertion elements (or IS elements). DNA segments between two IS elements mobilize intervening genes as a single unit. Composite transposons therefore have the ability to capture and mobilize random pieces of DNA from many diverse genetic locations, promoting genetic diversity. In relation to evolution of MDR, composite transposons can allow the movement of clustered antibiotic resistance genes from one bacterial genome to another through the capture of such genes into bacteriophages or conjugative plasmids that can then move readily between bacteria. Tn6026 is an example of a composite transposon, which consists of two independently mobile composite transposons, Tn6029 and Tn4352 (Cain et al., 2010; Martinez et al., 2013). Collectively the CRL (described above as Tn6026) confers resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole and trimethoprim. Similar structures, consisting of Tn6029 derivatives and essentially consisting of the same group of resistance genes have been described in plasmids such as pASL01a, which circulates in commensal E. coli in the human population in West Africa (Labar et al., 2012), and pHCM1, from a human E. coli isolate from a patient living in the Mekong Delta in Vietnam (Parkhill et al., 2001). However, the group of plasmids described by us, pO26-CRL, pO26-CRL₁₂₅, and pO111-CRL_115, is a clear example of plasmid mediated LGT of CRL between animal and human reservoirs in recent times.

IncA/C plasmids can provide conjugative functions in trans to mobilize integrative elements such as Salmonella Genomic Island 1 (SGI1) (Douard et al., 2010). SGI1 harbors genes encoding resistance to seven antibiotics (ampicillin, chloramphenicol, florphenicol, streptomycin, spectinomycin, tetracycline and sulphonamides). The resistance genes are clustered on a 13-kb class 1 integron, In104, embedded within a 42.4-kb genomic island embedded between thdF and yidY genes (Boyd et al., 2001; Levings et al., 2005). SGI1 is widely dispersed among a range of S. enterica serovars and Proteus mirabilus (Levings et al., 2005, 2006, 2007; Djordjevic et al., 2009; Le Hello et al., 2011, 2012), clearly indicating the lateral transfer of the element within bacteria occupying disparate hosts. A structurally similar CRL is also found in SGI2 described initially from a S. enterica serovar Emek strain in Australia. Major differences between SGI1 and SGI2 are in the physical location of the class 1 integron on the genomic backbone and in the different set of resistance gene cassettes seen associated with the integron found in SGI2. The integron, known as InEmek is a close variant of In104 seen in SGI1 (Levings et al., 2008) and the entire island shows characteristic features that mobilize SGI1.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.