Snapshot Study of Whole Genome Sequences of Escherichia coli from Healthy Companion Animals, Livestock, Wildlife, Humans and Food in Italy

Animals, humans and food are all interconnected sources of antimicrobial resistance (AMR), allowing extensive and rapid exchange of AMR bacteria and genes. Whole genome sequencing (WGS) was used to characterize 279 Escherichia coli isolates obtained from animals (livestock, companion animals, wildlife), food and humans in Italy. E. coli predominantly belonged to commensal phylogroups B1 (46.6%) and A (29%) using the original Clermont criteria. One hundred and thirty-six sequence types (STs) were observed, including different pandemic (ST69, ST95, ST131) and emerging (ST10, ST23, ST58, ST117, ST405, ST648) extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Eight antimicrobial resistance genes (ARGs) and five chromosomal mutations conferring resistance to highest priority critically important antimicrobials (HP-CIAs) were identified (qnrS1, qnrB19, mcr-1, blaCTX-M1,15,55, blaCMY-2, gyrA/parC/parE, ampC and pmrB). Twenty-two class 1 integron arrangements in 34 strains were characterized and 11 ARGs were designated as intI1 related gene cassettes (aadA1, aadA2, aadA5, aad23, ant2_Ia, dfrA1, dfrA7, dfrA14, dfrA12, dfrA17, cmlA1). Notably, most intI1 positive strains belonged to rabbit (38%) and poultry (24%) sources. Three rabbit samples carried the mcr-1 colistin resistance gene in association with IS6 family insertion elements. Poultry meat harbored some of the most prominent ExPEC STs, including ST131, ST69, ST10, ST23, and ST117. Wildlife showed a high average number of virulence-associated genes (VAGs) (mean = 10), mostly associated with an ExPEC pathotype and some predominant ExPEC lineages (ST23, ST117, ST648) were identified.


Introduction
Antimicrobial resistance has been recognized as one of the world's most pressing public health problems, with implications for human and veterinary medicine, wildlife and environmental ecosystems. In recent decades we have witnessed a dramatic spread and diffusion of multidrug resistant (MDR) pathogens. In the context of human medicine, the decrease in bacterial susceptibility to critically important antimicrobials (CIAs) and the quick diffusion of extended-spectrum beta lactamase (ESBL) producers are a major concern [1]. though some exhibited clonality including beef, swine and poultry with eight ST847, six ST10 and five ST117, respectively.
One hundred and fifty-eight different serotypes were predicted for the 279 strains analyzed. Forty-four strains were O nontypable with 20 different H types, meanwhile for one O107 isolate, the H type was not determined. Serotype variability was pronounced among sources, except for human sources with six O1:H7 strains (4, ST59; 2, ST95).
Phylosift analysis produced a maximum-likelihood tree ( Figure 1) with the first major split separating phylogroups B2 and D from A and B1. Tree topology was highly congruent with Achtman MLST and generally congruent with phylogroup distribution. Clade 1 was composed of two subclades, one mostly comprising phylogroup B2 and one primarily phylogroup D. ST69 was the prevalent sequence type of clade 1 with eight strains. Clade 2 was split into four subclades, the first mostly containing phylogroup D, the second and third were primarily phylogroup A and the fourth was dominated by phylogroup B1. The most common lineage of clade 2 was ST10 (16) all belonging to subclade 2. Strains of the same source clustered on distinct branches in some cases. However, sources were generally distributed across multiple clades.
Following de novo assembly, we identified 44 strains with scaffolds carrying a complete intI1 gene. Thiry-four of 44 strains carried cassette array genes and were annotated in order to characterize the different integron structures present in the collection. Twenty-three arrangements were characterized and designated letters (A-W). Derivatives of (A-W) were named with the letter of the principal structure followed by a number (Figure 6). Eleven ARGs were identified as intI1 related gene cassettes, namely those conferring resistance to aminoglycosides; aadA1, aadA2, aadA5, aad23, ant2_Ia, trimethoprim; dfrA1, dfrA7, dfrA12, dfrA14, dfrA17, and chloramphenicol; cmlA1. The most common integron cassette array was aadA1-dfrA1, present in 14/35 (40%) strains. Both sul1 and sul3 were identified in the 3'-CS of characterized integrons. Two isolates (one human and one poultry strains) harbored both intI1 and intI2 integrase genes.

Biocide Resistance Genes
Our collection was screened for different efflux pump genes and related regulators linked with the extrusion of a wide variety of molecules, including biocides and antimicrobials. Most genetic determinants, known to be chromosomally encoded, were widespread. Genes involved in quaternary ammonium compound resistance (emrE, mdfA), oxidative stress tolerance and protection/peroxygens resistance (ibpA, ibpB, sodA, sodB, ydeI, ymgB) and phenolic compound resistance (acrAB, fabI) were frequently identified. Integron-associated qacE and qacI genes were mostly identified in rabbit (17) and poultry (10) sources, alone or in combination ( Figure 2).
Complete VAG, ARG, MGE and efflux pump/biocide resistant gene carriage data are available in Supplementary Materials (Tables S1, S3 and S4).

Discussion
Using WGS we characterized 279 E. coli recovered from diverse animal and food sources in Italy to garner insight into the diversity of E. coli sequence types and the antimicrobial resistance and virulence genes they carry. We observed 136 E. coli STs, including pandemic (ST69, ST95, ST131) and emerging (ST10, ST23, ST58, ST117, ST405, ST648) EXPEC lineages. Most of the strains did not carry class 1 integrons but intI1 + isolates carried more ARGs compared to those that were intI1 − . Class 1 integron structure analysis among 50/279 (17.9%) intI1 + strains identified 22 class 1 integron variants among 34 structures. Limitations of short read sequence data precluded analysis of the remaining 16 class 1 integrons. ARGs that conferred resistance to HP-CIAs included qnrS1, qnrB19, mcr-1, bla CTX-M1,15,55 , and bla CMY-2 . Chromosomal mutations in gyrA/parC/parE were notable in poultry and rabbit sources.

Antimicrobial Resistance
The most common ARGs in all sources were tet (mostly tetA, 57/279), sul (mostly sul2, 45/279) and bla TEM (mostly bla TEM-1b , 43/279). These genes are generally reflective of common phenotypic resistance profiles of commensal E. coli of animal, food and human origin previously reported in Europe [12,18,[32][33][34][35][36]. The tet-sul-bla TEM genetic profile in livestock (cattle, swine, poultry, rabbit) reflects resistance to the most commonly used antimicrobials in Italian livestock production [37]. This profile was also observed in environments not commonly associated with direct antimicrobial selective pressure, like aquaculture (molluscs and fish), vegetables and wildlife. Resistance to tetracycline, sulfonamides and beta-lactams has been reported in aquaculture [35,38] and vegetables [36,39,40] in Europe. However, Italian data about AMR are lacking, probably due to the rare or absent antimicrobial use in these sectors [41]. Hence, a proper comparison between our findings and previous phenotypic reports in these sources is difficult to perform. A similar phenotypic AMR profile has been previously reported in wildlife [42,43], considered an AMR bioindicator [42,44,45] primarily because related AMR is strictly influenced by livestock and human density/activity [46,47].
The consistency of AMR profile identified in aquaculture, vegetables and wildlife with those observed in livestock, humans and companion animals supports AMR diffusion from settings with high antimicrobial use. Fecal contamination is considered the major path for AMR bacteria, genes and antimicrobial residues diffusion [7,8,32,48,49]. Sewage and manure contaminate groundwater and aquatic systems. Therefore, irrigation water and manure-based fertilizers may act as carriers of AMR bacteria and genes, polluting agricultural production [36,50]. Similarly, sewage and runoff from land could be responsible for AMR observed in aquaculture systems [51][52][53].
Cephalosporins (third, fourth and fifth generation), polymyxins and quinolones are considered HP-CIAs, the last-line of treatment for serious human infections. ESBL and polymixin ARGs, and pmr chromosomal mutations were detected in low frequency among the collection and were mostly identified in food-producing animals and related food. Our findings are in accordance with low cephalosporin and colistin phenotypic resistance identified in E. coli from livestock and related meat in Europe [12,18,33,35,56,57]. Nonetheless, mcr-1 and mcr-2 colistin resistance genes have been reported in European E. coli from poultry in Romania [65] and poultry and swine in Spain [66].
Notably, most ESBL genes (2 bla CTX-M-1 genes in wild animal and dairy strains; 1 bla CTX-M-15 in a dairy strain; 1 bla CTX-M-55 in a human strain) were found in proximity to ISEcp1. ISEcp1 is a member of the IS1380 family and has previously been associated with ESBL genes [67,68]. A single copy of this IS element is able to mobilize downstream genes through transposition [69,70]. De novo assemblies revealed that mcr-1 was flanked by IS6 family members in five out of seven carriers. Although assembly scaffold breaks preclude definitive identification of these IS elements, read-mapping identified IS26, a member of the IS6 family, in these strains (three rabbits and two swine). IS26 plays an important role in the evolution and mobilization of ARGs worldwide. These findings suggest a possible involvement of IS26 in the spread of mcr-1. Further studies are necessary to establish the precise IS elements involved and their effective ability to transmit mcr-1.
Fluoroquinolones and quinolones are important antimicrobials used in both hospital and community settings, with only a minor role in companion and food-producing animals [37,58]. Contrastingly, they are reported as some of the most used antimicrobials in poultry and rabbit production systems in Europe [41] despite the European Medicines Agency (EMA) not supplying stratified sales data of veterinary antimicrobials by food-producing animal species [37]. In recent years, there has been a significant increase in fluoroquinolone resistance in human clinical E. coli in Europe, with the highest resistance rates observed in Italy [71]. On the contrary, low resistance to these antimicrobials has been usually observed among livestock in the EU, with the exception of poultry [12,33]. In our study, quinolone/fluoroquinolone resistance was mainly identified in poultry (13) and rabbit (15) sources and arose due to chromosomal mutations in gyrA/parC/parE genes. Our findings are in accordance with those previously reported in poultry [33] and rabbits [18] and point to fluoroquinolone usage in these sectors.
Heavy metal resistance genes merA and terA identified in the collection are known to be genetically linked to a variety of AMR genes on Tn21-and Tn1721-like transposons (merA) and large MDR IncHI2 plasmids (terA) [72,73]. This likely explains the observation that strains carrying merA carried more than three times as many ARGs on average (7.29 vs. 1.87) compared to the whole collection, whilst five rabbit isolates carrying both merA and terA had an average of 12.4 ARGs, more than six times the collection average. Environmental heavy metal contamination may, therefore, continue to select for these MDR strains even if antimicrobial use in the sector were to cease.

Virulence-Associated Genes
We identified a surprisingly high number of VAGs (in particular in poultry, rabbit and human niches) despite the fact that our study focused on commensal and environmental E. coli. Furthermore, genetic virulence patterns indicated the presence of potential ExPEC pathotypes. This is rather concerning since E. coli is the most common causative agent of urinary tract infection (UTI) and bloodstream infection (BSI) globally [74,75]. This assumption was strengthened by the identification of different sequence types, recognized as pandemic (ST69, ST95, ST131) [74] or emergent ExPEC lineages (ST10, ST23 ST58, ST117, ST405, ST648) [76]. qacE and qacI genes, known to be associated with class 1 integron structure, were identified primarily within intI1 + sequences [77][78][79][80]. qacE and qacI encode small multidrug resistance efflux pump family (SMR) proteins, conferring resistance to quaternary ammonium compounds (QACs), which are commonly used disinfectants in hospitals, healthcare facilities and the food industry [81]. Resistance nodulation division family (RND) efflux pump genes (acrAB, acrEF, acrD) were also widely identified in the collection. RND family efflux pumps are involved in clinically significant MDR [82] and may target a wide variety of molecules [82][83][84][85]. The identification of efflux pump genes could suggest potential phenotypic resistance, where antimicrobial determinants are not identified. However, this event is strictly related to efflux pump overexpression genotypes which we did not evaluate [82].

intI1 + Strains Carry more ARGs and VAGs
Class 1 integrons are a reliable proxy for multiple drug resistance gene carriage [86] and play an essential role in the dissemination and evolution of MDR Gram-negative bacteria. As expected, intI1 + strains carried more ARGs (mean = 8) than intI1 − isolates (mean = 1). A wide variety of antimicrobial genetic determinants have been identified as intI1 related gene cassettes [87]. In particular, nearly half of the characterized integrons (15/34; 44.1%) carried an aadA1-dfrA1 cassette array. This array is widespread in both commensal and clinical strains isolated from animal and food sources [88]. The most frequent MGEs associated with class 1 integrons in our collection were Tn21 and close relatives, identified in 26 of 35 (74.3%) intI1 + strains. Tn21 transposon and related variants (belonging to the Tn3 family) are globally disseminated and frequently involved in multiple ARGs and class 1 integron carriage in Enterobacteriaceae [72,89,90].
intI1 + strains carried a higher number of VAGs (mean = 14) compared with intI1 − strains (mean = 9), and often harbored F plasmid replicons. F incompatibility group members are recognized as the majority of virulence-associated plasmids in E. coli. They are known to carry different antimicrobial resistance determinants [91,92] and class 1 integrons [93], creating a concerning combination of virulence and antimicrobial resistance traits. Our findings suggest that ARGs and VAGs may be co-localised on F plasmids in our collection. Long read sequencing data is required to interrogate this hypothesis.

Concerning Sources
Rabbit, poultry and wildlife sources were notable for concerning antimicrobial resistance and virulence profiles. Rabbit represented the niche carrying the highest number of mcr-1 genes (3 strains from one animal and two meat samples) among our collection. A recent Italian investigation [94] reported polymyxin (colistin) as a widely used antimicrobial in rabbit farms even though two reports indicated colistin was not a common treatment in rabbit breeding systems [41,95]. Resistance to colistin has been previously reported in breeding rabbits in Europe, supporting the data from the third report [96]. To our knowledge, this is the first report of mcr-1 identified in rabbit meat products. This finding suggests rabbit farms and meat should be investigated as a potential reservoir of mcr-1 and as a vector for its transmission. Concerningly, eight rabbit strains with ST20 (4/23) and ST40 (4/23) also harbored extensive virulence profiles. These profiles displayed co-presence of both ExPEC and IPEC VAGs and did not correspond to the usual pathotype observed in these lineages. ST20 and ST40 are reported as human diarrheagenic pathogens [97], often producing Shiga toxins [98][99][100]. The emergence of new hybrid pathotypes could represent a serious threat to public health [101,102], as witnessed in several previous outbreaks [103,104]. Further studies are needed to better understand this phenomenon and the potential animal sources involved in hybrid pathotype evolution and diffusion.
Poultry source strains (11/25; 44%) belonged to some of the most prominent ExPEC sequence types (ST131, 1; ST69, 1; ST10, 1; ST23, 3; ST117; 5), all of which were isolated from poultry meat and are previously reported in this niche [25,[105][106][107]. ExPEC lineages carried the highest number of VAGs (mean = 22) among poultry source and a high number of ARGs (mean = 5). ST117, a known avian pathogenic extraintestinal E. coli (APEC) lineage and a human pathogen [25,106,108] was the most common sequence type among poultry source. Poultry meat is a frequently investigated source of ExPEC E. coli, which can be transmitted to consumers through food consumption [22]. Fecal contamination of poultry carcasses during slaughter could allow potential ExPEC diffusion through the food chain [109,110]. Our findings underline the role of poultry as a source of potential ExPEC lineages and the importance of related meat as an ExPEC carrier to humans.
Although wildlife is not as widely scrutinized as food animals for potential threats to human health, a relatively high number of VAGs (mostly associated to ExPEC pathotype) were observed in our wild animal (mean = 11) and wild boar (mean = 8) isolates, with 10/47 (21.3%) isolates carrying ≥15. Notably, four strains belonged to some of the predominant ExPEC lineages (2, ST23; 1, ST117; 1, ST648). The ST648 strain carried the highest number of VAGs (37) in the entire collection. ST648 has been isolated from wild birds [111], humans, surface water, fish, vegetables and companion animals [112,113], has been linked to disease in both human and animals (pets, horses and wildlife) (https://enterobase.warwick.ac.uk/, accessed 19/10/2019) and is frequently reported as an ESBL gene carrier [113,114].

Conclusions
Briefly, our study reaffirmed the role of food-producing animals as a reservoir of potential zoonotic pathogens, with variable antimicrobial and virulence traits among the sources investigated. In particular, rabbits and poultry represented the most concerning sources, carrying the highest number of ARGs and VAGs. Poultry was associated with potential ExPEC strains. Meanwhile, rabbits were a source of potential hybrid E. coli pathogens and carriers of E. coli with mcr-1.
It should be noted that our study has two important limitations. Firstly, the small sample size of each source (in particular of companion animals) prevented accurate quantitative comparison between antimicrobial and virulence profiles and the evaluation of possible ARGs and VAGs transmission routes between environments. Secondly, our data originated from short read sequencing analysis. Therefore, we could not determine the location of all ARGs identified and their association with MGEs. Moreover, we were not able to establish similarities/dissimilarities between F plasmids, potentially responsible for VAGs and ARGs carriage in our collection.
Despite these limitations, our study provided basic information about AMR and virulence determinants circulating in various environments in Italy. Further investigations can add a deeper understanding of AMR and virulence epidemiological traits in Gram-negative bacterial populations of different settings.

Sampling
In the period between November 2010 and May 2018 a total of 300 commensal E. coli were collected from 12 different food, animal and human sources (dairy, beef, wild boar, rabbit, poultry, swine, vegetable, fishery, mollusc, wild animal and human), mainly in the Emilia Romagna region of Italy.
Food samples (chicken, rabbit and swine meat products, vegetables and fish) were collected from major supermarkets located in the province of Bologna and from the educational abattoir of the Veterinary Sciences Department (University of Bologna), during slaughtering procedures (sponge of beef carcasses). Milk, cheese and milking system filter samples, collected in a previous study by our research group, were included in the project.
Among animal samples, feces (cat and dog) and cloacal swabs (poultry) were collected from healthy individuals, that had not received antimicrobials in the month prior to the collection. Wild boar diaphragm samples were supplied by Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Bologna chapter.
Collection of human samples were approved by the University of Bologna Bioethics Committee under internal protocol number 0252770. Human feces were collected from healthy volunteers that were approached for recruitment in person. These participants had not had any antimicrobial treatments in the month prior the collection.
All food, animal and human samples were carried to the laboratory in aseptic conditions and analyzed within two hours from the time of gathering.
IZSLER Bologna, Forlì and Reggio Emilia chapters contributed to the strain collection with 97 hypothetical E. coli, isolated from different animal sources (mollusc, wild animal, rabbit, beef, companion animal, swine). Among these isolates, 12 strains of companion animal origin were isolated from diseased animals and were therefore excluded from the study to avoid the possibility of including pathogens.
The final collection was therefore characterized by 25 E. coli for each source (except for companion animal, n = 13), for a total of 288 strains.
A comprehensive description of the strain collection is provided in Supplementary Material, Table S1.

Bacterial Isolation
Twenty g of food sample (in the case of carcass sponge, each one separately) and wild boar diaphragm were placed into sterile blender bags, diluted in 180 mL of sterile EC-Broth (Oxoid, Basington, UK) and macerated in a stomacher for 1 min. Samples were incubated overnight at 37 ± 1 • C. Fecal samples (1 g each) were diluted (1:10) in peptone water (Oxoid, Basington, UK) and homogenated by vortexing.
Ten µL of overnight culture in EC-Broth (Oxoid, Basington, UK) and 10 µL of feces solution (or directly in the case of the cloacal swabs) were streaked onto MacConkey (Oxoid, Basington, UK) and Levine (Oxoid, Basington, UK) agar plates and incubated for 18-24 h at 37 ± 1 • C. For all the samples, lactose fermenting colonies were collected and assessed for Gram stain and standard biochemical test (indole probe). E. coli ATCC 25,922 was used as a control strain.

DNA Extraction and Isolate Identification
Bacteria were grown from glycerol stocks on Tryptone Soya Agar (TSA) (Oxoid, Basington, UK) plate overnight at 37 ± 1 • C. Genomic DNA was extracted using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany), following the manufacturer's instruction.
The following amplification parameters were applied: initial denaturation at 94 • C for 3 min, 30 cycles of denaturation at 94 • C for 30 s, annealing at 58 • C for 25 s, elongation at 72 • C for 30 s and a final extension at 72 • C for 3 min.
The amplified products were loaded onto a 2% agarose gel containing Syber Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA) and run in 1X TBE buffer at 100

WGS and Assembly
Library preparation was performed using the Nextera Flex library preparation kit (Illumina, San Diego, CA, USA). Briefly, genomic DNA was quantitatively assessed using an Invitrogen Quant-iT picogreen dsDNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The sample was then normalized to a concentration of 1 ng/µL and 10 ng of DNA was used for library preparation. After the tagmentation step, DNA was amplified with 12 PCR cycles using the facility's custom designed i7 and i5 barcodes as previously described [59].
Due to the number of samples, the quality control for the samples was done by sequencing a pool of samples using MiSeq V2 nano kit-300 cycles (Illumina, San Diego, CA, USA). Briefly, 3 µL of each library was pooled into a library pool, cleaned up using SPRI beads following the Nextera Flex clean up and size selection protocol. The pool was then sequenced using MiSeq V2 nano kit (Illumina, San Diego, CA, USA). Based on the sequencing data generated, the read count for each sample was used to pool libraries at a different amount to ensure equal representation in the final pool and to discard failed libraries (i.e., libraries with less than 100 reads). The final pool was then sequenced on Illumina NextSeq 500, 2 × 150 bp at Ramaciotti Centre for Genomics (University of New South Wales, Australia).
Sequencing reads were deposited in the National Center for Biotechnology Information (NCBI) database with study accession number PRJNA528851. Accession numbers for each sample are listed in Supplementary Material, Table S1.
Other sequences of interest (insertion sequence elements, AMR and virulence associated gene sequences), available at https://github.com/maxlcummins/E_coli_customDB and not present within the previous databases were also screened. Moreover, different efflux pump/biocide resistance gene sequences collected from GenBank were considered (Supplementary Material, Table S2). Pointfinder [121] was used to establish chromosomal mutation in gyrA/B-parA/C/E, ampC and pmrA/B, predicting phenotypic resistance to quinolones, AmpC-type cephalosporins and colistin, respectively.