CTX-M-producing Escherichia coli ST602 carrying a wide resistome in South American wild birds: Another pandemic clone of One Health concern

Wild birds have emerged as novel reservoirs and potential spreaders of antibiotic-resistant priority pathogens, being proposed as sentinels of anthropogenic activities related to the use of antimicrobial compounds. The aim of this study was to investigate the occurrence and genomic features of extended-spectrum β-lactamase (ESBL)-producing bacteria in wild birds in South America. In this regard, we have identified two ESBL (CTX-M-55 and CTX-M-65)-positive Escherichia coli (UNB7 and GP188 strains) colonizing Creamy-bellied Thrush (Turdus amaurochalinus) and Variable Hawk (Geranoaetus polyosoma) inhabiting synanthropic and wildlife environments from Brazil and Chile, respectively. Whole-genome sequence (WGS) analysis revealed that E. coli UNB7 and GP188 belonged to the globally disseminated clone ST602, carrying a wide resistome against antibiotics (β-lactams), heavy metals (arsenic, copper, mercury), disinfectants (quaternary ammonium compounds), and pesticides (glyphosate). Additionally, E. coli UNB7 and GP188 strains harbored virulence genes encoding hemolysin E, type II and III secretion systems, increased serum survival, adhesins and siderophores. SNP-based phylogenomic analysis, using an international genome database, revealed genomic relatedness (19–363 SNP differences) of GP188 with livestock and poultry strains, and genomic relatedness (61–318 differences) of UNB7 with environmental, human and livestock strains (Table S1), whereas phylogeographical analysis confirmed successful expansion of ST602 as a global clone of One Health concern. In summary, our results support that ESBL-producing E. coli ST602 harboring a wide resistome and virulome have begun colonizing wild birds in South America, highlighting a potential new reservoir of critical priority pathogens.


Introduction
Antibiotic resistance occurs naturally, however, the overuse and misuse of antibiotics in human and veterinary medicine, as well as in agriculture, livestock and animal husbandry have accelerated the process [1]. In addition to the selective pressure from antibiotics, resistance can also develop due to selective pressures from disinfectants (e.g. quaternary ammonium and triclosan), pesticides, and heavy metals, which are released into the environment by human activity [2].
Specifically, extended-spectrum β-lactamase (ESBL)-producing Enterobacterales have become an increasing public health issue worldwide, being recognized as critical priority pathogens by the World Health Organization [3]. Currently, ESBL-positive pathogens have been identified in companion and wild animals, becoming therefore a One Health problem [4,5]. ESBL enzymes confer resistance to both human and animal broad-spectrum cephalosporins. Among ESBLs, those of CTX-M family are the most widespread, and clinically relevant. Genes encoding ESBLs are often found on plasmids, which has enabled their spread, contributing to persistence and global dissemination of high-risk clones [6].
Noteworthy, wild animals have emerged as novel potential reservoirs and spreaders of antibiotic-resistant priority pathogens, since they have been directly exposed to polluted environments, and their feces are freely dispersed, possibly contaminating surface waters and soils [7]. While wildlife has been overlooked in the epidemiology of medically important antibiotic-resistant bacteria, isolation of ESBL-producing Escherichia coli from wild birds has begun to be reported worldwide, deserving epidemiological attention [7,8].
We hereby report microbiological and genomic characteristics of ESBL-producing E. coli colonizing wild birds in Brazil and Chile, highlighting its potential as spreaders of CTX-M genes in South America. In this regard, resistome (antibiotics, heavy metals, pesticides, and disinfectants), virulome and clonal relatedness have been investigated in depth.

Phylogenomic analysis
Genome assemblies of 266 E. coli strains belonging to ST602 and their metadata were retrieved from the Escherichia/Shigella Enterobase database (https://enterobase.warwick.ac.uk). ABRicate 1.0.1 (https:// github.com/tseemann/abricate) was used with CGE Resfinder 4.1 database (https://bitbucket.org/genomicepidemiology/resfinder) for screening of antimicrobial resistance genes in the 266 publicly available retrieved genomes, the two genomes obtained in our study (UNB7 and GP188), and an additional genome obtained from an E. coli ST602 (Pk-12 strain) isolated from an Eurasian coot Fulica atra, in Pakistan. Identity and coverage threshold were set to 90% and 95%, respectively. CSI Phylogeny 1.4 (https://cge.cbs.dtu.dk/services/CSIPhylogeny) was used with default settings to build an approximately maximumlikelihood phylogenetic tree of UNB7, GP188 and Pk-12 strains, along to the 266 genome assemblies retrieved from Enterobase. The genome of E. coli HB-Coli0 strain (ST602) was used as reference (RefSeq assembly accession: GCF_002116715.2), and iTOL (https://itol.embl.de) was used to midpoint rooting the generated tree, to annotate the tree with Enterobase metadata, and to delete from the tree strains that lacked country and/or source of isolation in Enterobase metadata. iTOL was also used to build heatmaps indicating presence/absence of resistance genes for each antimicrobial class based on data generated by ABRicate and Resfinder 4.1 phenotype predictions, as well as presence/absence of resistance genes found by ABRicate in genomes of strains inside clades containing isolates from wild birds (including UNB7, GP188 and Pk-12 strains).

Results
Among 118 cloacal swabs obtained from wild birds, two ceftriaxoneresistant E. coli strains (UNB7 and GP188) were isolated from a Creamybellied Thrush (Turdus amaurochalinus) and a Variable hawk (Geranoaetus polyosoma) ( Table 1). The Creamy-bellied Thrush was captured in a university campus in Brasilia, Midwest Brazil, whereas the Variable hawk was captured near the Andean mountain range in Chillán, Chile.
E. coli UNB7 exhibited resistance to ampicillin, cephalothin, cefotaxime, ceftiofur and ceftazidime, whereas E. coli GP188 displayed resistance to ampicillin, cephalothin, cefotaxime, ceftiofur, ceftazidime, nalidixic acid, ciprofloxacin, tobramycin, and chloramphenicol. Both isolates were ESBL producers, and belonged to sequence type ST602 (clonal complex CC446). Further genomic analysis of UNB7 revealed the presence of the bla CTX-M-55 ESBL gene and IncF-type, IncN, and IncX plasmids, whereas GP188 carried the bla CTX-M-65 gene and IncF-type and IncI1 plasmids. In addition to antibiotic resistance, E. coli strains displayed a wide virulome and resistome against disinfectants, heavy metals, and herbicides (Table 1 and Table 2).
Comparative phylogenetic analysis clustered UNB7 with human (318 SNPs difference), livestock (220 SNPs difference), and environmental (61 SNPs difference) E. coli strains of ST602, identified in China, USA, and Japan, respectively. Moreover, while GP188 strain was closest related (15 SNPs difference) to an E. coli strain isolated from a Chilean Andean condor, SNP differences with poultry strains from Ecuador and United States of America (USA) and a livestock strain from China, ranged from 106 to 385 SNPs (Fig. 1, Table S1)

Discussion
In this study, we identified two E. coli strains producing CTX-M-55 or CTX-M-65 ESBLs, in wild birds from South America. In this regard, CTX-M-55-positive E. coli have been mostly identified in human and animal hosts from Asian countries [12,13], and less frequently from European and North American countries [14][15][16][17][18]. In South America, CTX-M-55producing E. coli have been identified in human host, poultry, peri-urban wild animals and water samples, in Ecuador and Brazil [19][20][21][22]. CTX-M-65-producing E. coli have been mostly reported in human and animal hosts from Asian countries, mainly China and Korea [23][24][25][26], whereas in Europe, North America, and Oceania there are fewer reports restricted to human hosts [27][28][29]. In South America, E. coli carrying CTX-M-65 ESBL genes have been identified in humans and wild bids in Bolivia and Chile, respectively, and in a giant anteater in a zoo, in Brazil [30][31][32][33].
The MLST analysis showed that both CTX-M-55-and CTX-M-65positive E. coli strains belonged to ST602 (CC446). This clone has been reported globally in humans, pets, wild and food-producing animals, and water and food samples. Specifically  Table S2).
Phylogenetic analysis and comparative resistome from wild birds suggest that E. coli ST602 producing carrying CTX-M enzymes have been circulating in wild birds in Brazil, Chile, Australia, and Pakistan at least since 2017 (Fig. 1B). On the other hand, genomic relatedness between the CTX-M-65-positive E. coli strain GP188 and another CTX-M-65positive E. coli strain (DF391), previously identified in Chile, was confirmed. Moreover, both isolates were clustered with four poultry E. coli isolates from Ecuador and USA, and a livestock isolate from China.
The CTX-M-55-producing E. coli strain UBN7 was closely related to an environmental isolate from Japan, being further clustered with a livestock strain from USA, and a human isolate from China. Although, the CTX-M-27-positive E. coli ST602, isolated from a Seagull in Australia, was not isolated in this study, our phylogenetic analysis also highlights the genomic relatedness with environmental, human, and food CTX-M-27-producing E. coli strains identified in Japan, China, and Cambodia, respectively.
Noteworthy, both strains identified in our study displayed a wide resistome against disinfectants, heavy metals, and herbicides. In this respect, the operon phn, which confers resistance to glyphosate (an herbicide largely used in agriculture, silviculture, and urban gardens) was identified. Although some bacteria, such as Achromobacter spp., Ochrobactrum anthropi, Sinorhizobium meliloti, Rhizobium radiobacter, and Burkholderia pseudomallei, utilize glyphosate as a source of phosphorus, E. coli is unable to use this herbicide as an inorganic phosphate (P i ) source [34]. Therefore, the presence of the operon phn in UNB7 and GP188 could suggest an adaptative tolerance mechanism to pesticides. In fact, the wide resistome could be related to environmental pollution by anthropogenic activities, since agricultural and industrial activities, including fertilizer application and mining, have contributed to heavy metal and herbicide accumulation in the environment [35].
E. coli strains UBN7 and GP188 also carried genes conferring tolerance to arsenic, antimony, cadmium, cobalt, copper, chromium, iron, magnesium, mercury, zinc, tellurite, tungsten, nickel, silver, and molybdopterin. Metal pollutants can be released into the environment from many sources, such as agriculture, battery recycling, and metal production processes, as they resist to degradation can persist in water and soil [36]. Specifically, heavy metal contamination has been related with co-selection of other antimicrobial resistance genes and potentially contributes to the spread of antibiotic resistance [37][38][39]. Additionally, both isolates carried genes conferring tolerance to quaternary ammonium compounds (i. e. emrDK, mdtEFKN, acrEF, tehAB). Therefore, the extensive use of disinfectants in industry, hospitals, domestic households, and cosmetic products may be imposing a selective pressure [40].
As a limitation of this study, a relatively small number of wild birds were sampled, and it was not possible to determine exactly how these animals acquired ESBL-producing E. coli. Unfortunately, there is a lack of information regarding the environmental factors and mechanisms that facilitate the transmission of E. coli strains from wildlife environments to synanthropic environments. However, it is well-known that E. coli is normally found in the intestinal tract of vertebrates, being widely used as an indicator of faecal contamination of food and water [41]. Therefore, transmission of antimicrobial-resistant E. coli from anthropogenically polluted environments to wildlife environments can occur dynamically and continuously. Although, antibiotic-resistant E. coli have been reported in wild birds at least since 1978 [42], it is not clear how ESBL-producing E. coli make their way into the wildlife environment. Most likely, ESBL-positive E. coli can reach the environment from hospital and/or community pollution [41,[43][44][45][46], whereas acquisition of antimicrobial-resistant bacteria by wildlife is probably mediated by horizontal gene transfer on conjugative plasmids, from clinical isolates, or from the intake of resistant bacteria from aquatic environments polluted by industrial, agricultural and domestic waste [44,45,47].

Conclusions
In conclusion, we report the identification and genomic features of two ESBL (CTX-M-55 and 65)-producing E. coli colonizing wild birds in countries with endemic occurrence of human infections caused by CTX-M producers, highlighting new potential reservoirs of critical priority pathogens. E. coli strains belonged to ST602, a lineage of global distribution. Worryingly, our epidemiological tracking revealed global dissemination of this clone at the human-animal-environment interface. Additionally, we report that ST602 isolated from wild bird species has been harboring CTX-M enzymes at least since 2017. Specifically, the wide resistome of CTX-M-55 and CTX-M-65-positive E. coli strains ST602, for clinically relevant cephalosporins, disinfectants, heavy metals, and herbicides, could denote environmental pollution by anthropogenic activities related to the use of these antimicrobial and biocides compounds. Therefore, these data provide important information to be used in epidemiological studies of critical ESBL-producing pathogens within a One Health perspective, as well as to understand genomic aspects related to adaptation and dissemination of critical priority pathogens at the human-animal-wildlife interface. Hence, we strongly encourage continuous surveillance of ESBL-producing E. coli in wild birds in Latin America for a better comprehension of the transmission pathways and clinical impacts of such pathogens in wildlife populations.

Accession numbers
The datasets presented in this study can be found in online repositories. Both GP188 and UNB7 Genome shotgun projects have been deposited at DDBJ/ENA/GenBank under the accession JAJNMH000000000 and JAAVSK000000000, respectively. Additionally, genomic and epidemiological information of both E. coli strains have been deposited at OneBR (EcBr) platform (http://onehealthbr. com), under IDs ONE133 (GP188) and ONE10 (UNB7), respectively.  Table S2).

Author contributions
GD, DF, EM and NL designed the experiments. DF, BM, LS, and LP performed the sampling campaign. GD, DF, BC, FE and QM performed the experiments. GD, HF, EP and DF performed the WGS and Phylogenetic analyses and images. GD prepared the manuscript. All authors discussed the results, reviewed and edited the manuscript, and read and approved the final version of the manuscript.

Funding
This study was supported by the Bill and Melinda Gates Foundation (Grand Challenges Explorations Brazil OPP1193112). Under the grant conditions of the Foundation, a CC BY or equivalent license is applied to the author accepted manuscript version arising from this submission. Additionally, this study was supported by the Fundação de Amparo à