Colistin-Resistant mcr-1-Positive Escherichia coli ST131-H22 Carrying blaCTX–M–15 and qnrB19 in Agricultural Soil

The pandemic Escherichia coli sequence type 131 (ST131) carrying plasmid-mediated colistin resistance mcr genes has emerged worldwide causing extraintestinal infections, with lineages belonging to three major clades (A, B, and C). Clade B is the most prevalent in animals, contaminating associated meat products, and can be transmitted zoonotically. However, the blaCTX–M–15 gene has only been associated with C2 subclade so far. In this study, we performed a genomic investigation of an E. coli (strain S802) isolated from a kale crop in Brazil, which exhibited a multidrug-resistant (MDR) profile to clinically significant antimicrobials (i.e., polymyxin, broad-spectrum cephalosporins, aminoglycosides, and fluoroquinolones). Whole-genome sequencing analysis revealed that the S802 strain belonged to serotype O25:H4, ST131/CC131, phylogenetic group B2, and virotype D5. Furthermore, S802 carried the clade B-associated fimH22 allele, genes encoding resistance to clinically important antimicrobials, metals, and biocides, and was phylogenetically related to human, avian, and swine ST131-H22 strains. Additionally, IncHI2-IncQ1, IncF [F2:A-:B1], and ColE1-like plasmids were identified harboring mcr-1.1, blaCTX–M–15, and qnrB19, respectively. The emergence of the E. coli ST131-H22 sublineage carrying mcr-1.1, blaCTX–M–15, and qnrB19 in agricultural soil represents a threat to food and environmental safety. Therefore, a One Health approach to genomic surveillance studies is required to effectively detect and limit the spread of antimicrobial-resistant bacteria and their resistance genes.


INTRODUCTION
The rapid spread of plasmid-mediated colistin resistance mcr genes has gained worldwide attention as a critical public health issue, since colistin is a last resort antimicrobial used to treat severe infections caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria (Perez et al., 2016;Tsuji et al., 2019). Currently, epidemiological studies have shown that the spread of colistin-resistant mcr-positive bacteria is not a concern restricted to hospitals, but also represents a growing problem involving environmental and food safety. In this regard, various factors such as environmental sources, food-producing animals, international travel, and food trade, have accelerated the worldwide spread of mcr-type genes at the human-animalenvironment interface (Liu et al., 2016;Hassan and Kassem, 2020;Johura et al., 2020).
Specifically, contamination of crops by critical priority pathogens is of great concern, since these pathogens can also contaminate vegetables for consumption (Cantas et al., 2013;Araújo et al., 2017;Lopes et al., 2017;Reid et al., 2020;Lopes et al., 2021), increasing the risk of human exposure to antimicrobial-resistant bacteria, including mcr-positive strains. Despite this, little is known about the occurrence of bacteria carrying mcr-type genes in soils. Therefore, in this study, we performed a genomic investigation of an mcr-1-positive E. coli strain exhibiting an MDR profile to clinically significant antimicrobials and isolated from agricultural soil in the light of the One Health context that integrates human, animal, and environmental health.

Soil Sampling and Bacterial Isolation
During a surveillance study conducted between October and December 2019 to monitor the presence of clinically significant MDR Gram-negative bacteria in crops, 15 soil samples with a history of cow manure use were collected at a depth of ∼5 cm from chicory (n = 3), kale (n = 3), mustard (n = 3), parsley (n = 3), and chive (n = 3) crops on a farm in the state of São Paulo (21 • 00 36.0 S; 47 • 27 00.0 W), Brazil. All samples were stored at 4 • C and processed within 24 h. For bacterial isolation, 1 g of soil was inoculated in Luria-Bertani broth (Oxoid Ltd., United Kingdom) and incubated at 37 • C for 24 h. Subsequently, the cultures were streaked onto MacConkey agar plates (Oxoid Ltd., United Kingdom) supplemented with ceftriaxone (2 µg/ml) or colistin (2 µg/ml). Colonies were picked from the selective plates, subcultured, and streaked to obtain pure cultures. Bacterial identification was initially performed using 16S rRNA gene sequencing (Weisburg et al., 1991).

DNA Isolation and Whole-Genome Sequencing
For whole-genome sequencing (WGS), total DNA was extracted from an overnight culture using the GenElute TM Bacterial Genomic DNA Kit (Sigma-Aldrich, United States) according to the manufacturer's instructions. Sequencing was performed using the Illumina HiSeq 4000 (2 × 150 bp) platform (Illumina, United States).

Phylogenetic Analysis
For phylogenetic analysis, we selected the E. coli strain reported in this study and 849 other strains representative of all clades 1 www.eucast.org 2 http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 3 http://clermontyping.iame-research.center/ (A, B, and C) of E. coli ST131. A minimum spanning tree was constructed based on the MSTree v.2 algorithm and the wgMLST scheme in Enterobase 4 . The tree was visualized with iTOL v.5.7 (Letunic and Bork, 2019).

Conjugation Assays
Conjugation assays were conducted using azide-resistant E. coli C600 as recipient strain. Overnight cultures of donor and recipient strains were mixed (ratio 1:1) and incubated for 18 h at 37 • C without shaking as previously described (Furlan et al., 2020a). Transconjugants were selected using MacConkey agar (Oxoid Ltd., United Kingdom) supplemented with sodium azide (200 µg/ml) and ceftriaxone (2 µg/ml), or sodium azide (200 µg/ml) and colistin (2 µg/ml), and confirmed by PCR for the detection of mcr-and bla CTX−M -type genes as described above.

MDR mcr-1-Positive ESBL-Producing E. coli Isolated From Agricultural Soil
In this study, the presence of a mcr-1-positive ESBL-producing E. coli strain (named S802), identified by 16S rRNA gene sequence analysis and pairwise genome comparison of average nucleotide identity, was confirmed in one soil sample from the kale crop. In addition, the E. coli strain S802 displayed an MDR profile, defined as resistant to at least one antimicrobial of three or more different categories (Magiorakos et al., 2012). The MDR profile of E. coli S802 included resistance to colistin, penicillin, cephalosporins, aztreonam, aminoglycosides, quinolones, tetracycline, and chloramphenicol. In contrast, the strain displayed an intermediary resistance profile to ampicillin/sulbactam, remaining susceptible to piperacillin/tazobactam, amikacin, and carbapenems ( Table 1).

Identification of the Pandemic Escherichia coli ST131 Lineage and Phylogenetic Analysis
WGS revealed that E. coli strain S802 belonged to serotype O25:H4 and phylogroup B2, known for including highly virulent extraintestinal lineages. Strain S802 carried fimH22 allele and 4 https://enterobase.warwick.ac.uk/species/index/ecoli was assigned to the clade B pandemic ST131/CC131 lineage (Figure 1). In addition, the clonotype CH40-22 was determined. Phylogenetic relatedness among 850 genomes of globally reported E. coli ST131 strains (Figure 2A) assigned S802 to a cluster comprising human E. coli ST131-H22 strains from Spain, Netherlands, Germany, and Belgium; one avian strain from Germany; and one swine strain from Spain. E. coli strain S802 was most related to two strains isolated from humans in Spain in 2010 ( Figure 2B).
The pS802-QnrB was a small 3,041 bp ColE1-like plasmid, containing 52.09% GC and only the qnrB19 and pspF (truncated transcription activator) genes ( Table 2). The qnrB19 gene was located in the conserved genetic context between the sequence encoding the regulatory RNAII and the Xermediated recombination site. The pS802-QnrB plasmid was related to other ColE1-like plasmids of Enterobacterales isolated worldwide at the human-animal-environment interface and shared 70% query coverage and ∼99.5% nucleotide identity with plasmids of the same incompatibility group of E. coli strains isolated from poultry in Brazil (GenBank accession numbers: KX452393.1 and KX452394.1), similarly to the IncHI2-IncQ1 plasmid in this study.

DISCUSSION
The emergence of clinically relevant bacterial strains in soils is an underestimated public and environmental health problem that requires attention. In this regard, mcr-positive E. coli lineages from farming soil and agricultural soil have been previously reported in China (Zheng et al., 2017) and Algeria FIGURE 4 | Comparison between IncHI2 plasmids. Plasmids of E. coli strains: plasmid unnamed (GenBank: CP015833.1) from a human in the United Kingdom; plasmid: 1 (GenBank: LR882919.1) from a human in the Netherlands; pECJS-B60-267 (GenBank: KX254341.1) from a pig in China; pS802-MCR (GenBank: MW495059.1) from soil in Brazil; pMCR_915_E1 (GenBank: MT929285.1), pMCR_1085_C1 (GenBank: MT929286.1), pMCR_1139_D1 (GenBank: MT929287.1), and pMCR_170_D1 (GenBank: MT929288.1) from turkey meat in the Czech Republic. Plasmids of Salmonella Schwarzengrund strains: p280_12888 (GenBank: CP045449.1) and p280_9355 (GenBank: CP045446.1) from poultry in Brazil. Matches with less than 70% identity and no matches appear as blank spaces. Resistance genes to antimicrobials/biocides and metals are indicated in red and blue, respectively. FIGURE 5 | Comparison between IncF plasmids. Plasmids of E. coli strains: pAMPD02 (GenBank: CP058310.1) from a giant panda in China; 134q (GenBank: CP023363.1) from a canine in Scotland; pSDJ2009-52F (GenBank: MH195200.1) and pCERC4 (GenBank: KU578032.1) from a human in Australia; plasmid unnamed (GenBank: CP027485.1) from a human in the United States; pL65-2 (GenBank: CP034739.1) from a goose in China; p13P484A-2 (GenBank: CP019282.1) from a pig in China; pVPS18EC0676-1 (GenBank: CP063726.1) from veal in the United States; pS802-CTX-M (GenBank: MW495061.1) from soil in Brazil. Plasmid of Salmonella Kentucky: pCVM29188_146 (GenBank: CP001122.1) from poultry in the United States. Matches with less than 70% identity and no matches appear as blank spaces. Resistance genes to antimicrobials and metals are indicated in red and blue, respectively, whereas virulence genes are indicated in orange. (Touati et al., 2020), respectively. In Brazil, mcr-type genes from soil samples have only been detected in total DNA or cultivable microbiota so far (Oliveira et al., 2019;Furlan et al., 2020b;Dos Santos et al., 2020). In this study, we report for the first time the presence of an mcr-1-positive E. coli isolated from the soil ecosystem in American countries, representing a potential risk of human exposure to antimicrobial-resistant bacteria.
E. coli belonging to the ST131 pandemic high-risk clone has been identified in human, animal, environmental, and food samples (Figure 1). In addition, E. coli ST131 has been frequently reported carrying clinically significant antimicrobial resistance genes, such as mcr-types and/or ESBL genes (Rodrigues et al., 2017;Reid et al., 2019), and associated with extraintestinal diseases, mainly bloodstream and urinary tract infections (Liu et al., 2018;Mamani et al., 2019;Reid et al., 2019).
Additionally, pS802-CTX-M harbored the ColV region, frequently identified in avian pathogenic E. coli (APEC) and associated with increased fitness and virulence of these strains (Johnson et al., 2006). The presence of ColV plasmid in E. coli strains isolated from humans can indicate evidence of zoonotic transmission (Rodriguez-Siek et al., 2005;Liu et al., 2018). As detected in the present study, ColV plasmids can also carry multiple antimicrobial resistance genes, which is clinically relevant due to the combination of virulence and resistance determinants in a single mobile genetic element (Flament-Simon et al., 2020).
Although the origin of the E. coli ST131-H22 high-risk sublineage carrying the mcr-1.1, bla CTX−M−15 , and qnrB19 genes was not investigated, cow manure used for soil fertilization was the most likely source. In addition, other animal (e.g., wild animal feces), human (e.g., sewage), and environmental (e.g., contaminated irrigation water) sources could be involved in the dissemination of clinically relevant bacterial strains (Beuchat, 2002;Cantas et al., 2013;Araújo et al., 2017).
The range of hosts and sources of the E. coli ST131-H22 sublineage, including soil detected here, supports a genetic versatility and adaptation mediated by the gene content, which includes genes encoding resistance to antimicrobials, biocides, and heavy metals. In fact, the plasmids pS802-MCR and pS802-CTX-M co-harboring resistance genes to antimicrobials, biocides, and heavy metals were identified (Figures 3-5).
In this regard, heavy metals could come from sources such as contaminated irrigation water, inorganic fertilizers, and pesticides commonly used in agricultural practices, remaining in the environment for long periods (Gimeno-Garcia et al., 1996;Sipter et al., 2008;Osaili et al., 2016;Bhilwadikar et al., 2019). Consequently, these compounds, as well as biocides, may act as selectors of strains resistant to antimicrobials.
Finally, the presence of MDR pathogens displaying a broad resistome in agricultural soil could lead to contamination of vegetables and, since these foods are usually consumed raw, the risk of human exposure to antimicrobial-resistant bacteria with clinical interest increases (Reid et al., 2020;Lopes et al., 2021). Although ingestion of these bacteria may not immediately have a direct impact on health, colonization by this pathway can contribute to the horizontal gene transfer of antimicrobial resistance to the gut microbiome (Maeusli et al., 2020). Thereafter, a potential threat to human health would be associated with future endogenous infections, mainly in immunosuppressed patients, in whom therapeutic failure could occur.

CONCLUSION
The emergence of zoonosis-associated E. coli ST131-H22 carrying a broad resistome, including mcr-1.1, bla CTX−M−15 , and qnrB19, in agricultural soil represents a potential risk of human and animal exposure to antimicrobial-resistant bacteria and/or their resistance genes, posing a threat to public and environmental health. Also considering the possible contamination of vegetables for consumption from soil pathogens, appropriate measures, such as the improvement of agricultural practices, in addition to stricter regulations, need to be taken. Therefore, a One Health approach is required to effectively limit the spread of MDR bacteria and prevent their health impacts.

AUTHOR CONTRIBUTIONS
RL, JF, LS, and IG carried out the research. RL and JF performed data curation and formal analysis. RL, JF, and ES conceived and designed the study, and reviewed and edited the manuscript. RL drafted the original manuscript. ES coordinated and acquired funding for the study. All authors read and approved the final manuscript.