The intensive use of antimicrobials in human and veterinary medicine has resulted in an emergence of antimicrobial resistance (AMR) in humans, animals and the environment at large (Radhouani et al., 2014; Berendonk et al., 2015). Enterobacteriaceae producing ESBLs have increasingly emerged due to the widespread use of cephalosporins and represent a major challenge in infection control (Pitout and Laupland, 2008). Currently, the most commonly encountered ESBL enzyme is the plasmid-encoded CTX-M-type. In particular, an E. coli clone of sequence type 131 (ST131) carrying the CTX-M-15 ESBL has been commonly found in clinical and non-clinical settings (Nicolas-Chanoine et al., 2014).

Previous studies have suggested the environment including water, soil and wildlife as the source for clinically relevant ESBL-E. coli (Wright, 2010; Blaak et al., 2015; Guenther et al., 2017), thereby possibly transmitting certain ESBL-E. coli clonal lineages or ESBL-plasmids from natural environments to humans, livestock or companion animals. Wild migratory birds have been discussed as sentinels and a potential vectors for the transboundary spread of ESBL- producing bacteria (Raza et al., 2017). Furthermore, wildlife has been considered as reservoir of potentially zoonotic extra-intestinal pathogenic E. coli (ExPEC) strains in earlier studies (Ewers et al., 2009; Gordon and Cowling, 2012).

Recently, it has been suggested that certain clonal lineages distinguished by very low number of single nucleotide polymorphisms (SNPs) circulate at the human-animal-environment interfaces which strongly supports the One Health perspective of AMR (Falgenhauer et al., 2016; Schaufler et al., 2016). Pakistan is among the Asian countries that harbor a large number of migratory birds during winter migration along the Indus route coming from Siberia and Central Asia. In this study, we screened wild migratory birds from four different wetland habitats along the Indus migration route in Pakistan to assess the prevalence of ESBL-E. coli and to subsequently characterize them in-depth via whole genome sequencing to assess AMR genes, multi locus sequence types (MLST), plasmid replicon types, and virulence-associated genes (VAGs). Additionally, the core genomes of identical STs were analyzed for SNPs.

Wild migratory birds have been suggested as a reservoir of ESBL-producing E. coli in a number of studies worldwide (Guenther et al., 2011, 2012; Bonnedahl et al., 2015; Atterby et al., 2016). More recently, we reported the occurrence of bla-_CTX−M−15 producing Klebsiella pneumoniae (Raza et al., 2017) in wild migratory bird populations in Pakistan. We therefore also screened for ESBL-producing E. coli and their clonal relatedness using WGS, as there is lack of knowledge regarding genetic diversity of ESBL-E. coli isolates from environmental niches in Asia. E. coli is an excellent indicator species to study the spread of AMR through fecal pollution of water and waterfowl can be considered as sentinel of AMR in the environment (Guenther et al., 2011). The present study indicates high carriage rates of ESBL-producing E. coli (17%) in migratory birds along the Indus migration route in Pakistan. This high prevalence mirrors those reported in migratory gulls from Bangladesh (17.3%) (Hasan et al., 2014) and is comparable to another study from Bangladesh which reported 30% ESBL-E. coli from wild ducks (Hasan et al., 2012). This is underlining the important role of waterfowl as carrier of ESBL-producing E. coli in Asia and also adding the important Indus avian migration route to the environments influenced by human healthcare practices.

WGS showed bla_CTX−M−15 was the predominant ESBL genotype in this study. This is in agreement with some previous findings from wild birds in Bangladesh (Hasan et al., 2014), Germany (Guenther et al., 2010) and North America (Poirel et al., 2012). CTX-M-15 has now a worldwide distribution and although it is commonly associated with human and pet ESBL-isolates, it is also very common in avian wildlife (Wang et al., 2017).

In fact, summing up the current literature it becomes obvious that the emergence of ESBL-producing E. coli in wildlife is associated with the success of the bla_CTX−M family in hospitals (Guenther et al., 2011). The reason why bla_CTX−M producing E. coli are also very successful in the environment remain unclear but recent studies suggest that plasmids carrying those genes confer more advantages than mere resistance to the bacterial host strains (Schaufler et al., 2016). A previous study also indicated high rates of bla_CTX−M−15 from human clinical isolates in Pakistan (Habeeb et al., 2014), however as we did not include human isolates in this study their relatedness remains to be clarified in the future.

Besides their spread via plasmids, very recently the new trend of chromosomal integration of ESBL-encoding genes has been demonstrated in clinical E. coli isolates of ST38, ST410, ST131 and ST648 (Hirai et al., 2013; Rodríguez et al., 2014; Falgenhauer et al., 2016) and also in non-clinical ST38 isolates from wild birds (Guenther et al., 2017). Similarly, we detected the chromosomal insertion of bla_CTX−M−15 genes among E. coli of different STs (ST224, ST1722 and ST58), which have been found as plasmid carrying ESBL-producers in clinical and non-clinical samples, worldwide (Zurfluh et al., 2013; Leangapichart et al., 2016). This scenario has also been recently shown for E. coli strains of ST38 from Mongolian wild birds, which were very closely related to a clinical outbreak strain from the UK (Guenther et al., 2017).

As mentioned above, wildlife has been reported to carry ExPEC strains, we therefore also screened for the occurrence of VAGs to gain information on pathotype. However, we detected no ExPEC strain in our isolates. Most of the strains harbored only a few VAGs and are likely commensal strains. However, all the E. coli carried serum resistance ompA gene (Table 1). We also found high frequency of astA and iha genes. These are only putative virulence genes and their exact involvement in the pathogenesis is not well understood, although they have been frequently reported in enteroaggregative E. coli and avian pathogenic E. coli (Nowak et al., 2017).

We found a large diversity of sequence types within the avian isolates including typical ESBL-associated sequence types like ST10, ST224, ST617 (Guenther et al., 2011; Sherchan et al., 2015), and ST354 (Zhang et al., 2016). However, globally distributed high risk clones like ST131, ST410, and ST648 were not found in this study. Earlier studies from human clinical E. coli isolates from Pakistan reported those sequence types including ST131 and ST648 (Mushtaq et al., 2011; Pesesky et al., 2015), indicating that different clonal population of E. coli might be present in wild birds and the human population in Pakistan but this finding can also be due to the low number of birds sampled.

Interestingly we found identical STs in isolates originating from different avian host species and geographic locations (Figure 2). Core genome phylogenetic analysis of those isolates showed that within identical STs only a small number of SNPs ranged from 1 to 29 were found. This suggests a recent interspecies transmission and long-distance dissemination of certain clonal ESBL-lineages by wild birds as it has been reported earlier (Guenther et al., 2017). The origins of most of these birds are remote areas in Siberia and Central Asia and exposure to antimicrobials is less likely in these areas. The high rates of MDR isolates detected from the wild migratory bird are of concern and could be due to anthropogenic activities from the surrounding environment. In addition to the clonal spread of certain STs our data showed the common occurrence of a plasmid replicon type (IncFIB, F-:A-:B53) linked to a 130 kb plasmid. This plasmid was found in all four wetlands tested and in five of the seven different avian species. Together with the large number of minor STs points toward the spread of a bla_CTX−M resistance plasmid of the pMLST type F-:A-:B53 among a naive E. coli population in the avian hosts.

The transmission dynamics of ESBL-producing E. coli in a natural environment are complex. Wild birds have been suggested as sentinels for the spread and transmission of multi-resistant strains in the environment. It is widely believed that the spread of ESBL-E. coli is driven both by plasmid transfer in commensal and pathogenic strains as well as by the clonal spread of certain lineages in local areas. In this study we were able to detect both main mechanisms in wild migratory birds in Pakistan underlining the suitability of avian sentinels. In addition our data highlights the potential for regional and intercontinental transmission of ESBL-producing E. coli clones and resistance plasmids via migratory birds.

MM, SG: conceived and designed the experiments; MM, SR, and FS: collected the data and samples; MM, KS, NR, FS, and PS: performed laboratory analysis; SG, MM, SR, and TS: analyzed the data; TS and SG: performed WGS; MM and SG: wrote the article. All authors have read and approved the final draft of the manuscript.