Extended-spectrum β-lactamase-producing Escherichia coli isolated from raw vegetables in South Korea

The increasing prevalence of oxyimino-cephalosporin-resistant Enterobacteriaceae has become a global concern because of their clinical impact on both human and veterinary medicine. The present study determined the prevalence, antimicrobial susceptibility, and molecular genetic features of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) isolates from raw vegetables. A total of 1324 samples were collected from two agricultural wholesale markets in Incheon, South Korea in 2018. The ESBL-EC strains were isolated from 0.83% (11/1324) samples, and all of them were resistant to ampicillin, piperacillin, cefazoline, cefotaxime, and nalidixic acid and yielded CTX-M-type ESBL, including CTX-M-14, CTX-M-15, CTX-M-55, CTX-M-27, and CTX-M-65. The isolates belonged to phylogenetic subgroups D (n = 5), A (n = 4), and B1 (n = 2). Multilocus sequence typing revealed nine known E. coli sequence types (STs), including ST10, ST38, ST69, ST101, ST224, ST349, ST354, ST2509, ST2847, and two new STs. Notably, ST69, ST10, ST38, and ST354 belong to the major human-associated extraintestinal pathogenic E. coli lineages. Our results demonstrate that ESBL-producing multidrug-resistant pathogens may be transmitted to humans through the vegetable intake, highlighting the importance of resistance monitoring and intervention in the One Health perspective.


Discussion
Fresh produce is usually consumed raw or not properly heated. Consequently, a considerable portion of recent foodborne outbreaks has been attributed to fresh produce contaminated by pathogens 27 . Likewise, antimicrobial resistance can readily spread to humans through the vegetables contaminated with resistant bacteria. ESBL-ECs have also been found in vegetables, principally ready-to-eat vegetables 19,25 and raw vegetables [28][29][30][31][32] . In this study, we isolated the cefotaxime-resistant ESBL-ECs from eleven (0.83%) of 1324 raw vegetables, indicating a lower Figure 1. National wide distribution of cefotaxime-resistant E. coli isolates from vegetables. The black circle and green area represented the location of the wholesale market for agricultural products from which vegetables were collected and the production site of them from which resistance was detected, respectively. The map was generated by using software program Microsoft PowerPoint 2016. The prevalence was about four times higher in the stem-type (2.94%) than the leafy-type samples (0.68%), but no isolates were detected in fruit and root types. All 11 ESBL-ECs from vegetables in this study were MDR strains with resistance to ampicillin, piperacillin, cefazoline, cefotaxime, and nalidixic acid. Similarly, resistance to ampicillin, cefazoline, and cefotaxime were also reported on livestock and human ESBL-ECs in South Korea 8,22 . In addition, all of the vegetable isolates were susceptible to amikacin and tigecycline, which was consistent with previous reports in clinical isolates 33,34 .  E. coli phylogenetic groups A and B1 are typically commensal strains, whereas the B2 and D groups are mainly extraintestinal virulent strains 48 . In our study, the proportions were comparable between these commensal (6/11, 54.5%) and more virulent (5/11, 45.5%) lineages. MLST analysis revealed 11 different STs for the 11 ESBL-ECs in this study. Interestingly, four isolates (4/11, 36.4%) belonging to ST69, ST354, ST38, and ST10 were among the top 20 global extraintestinal pathogenic E. coli (ExPEC) lineages 49 , which are responsible for the majority of extraintestinal diseases, such as urinary tract infection, sepsis, and neonatal meningitis, and may have food animal sources 50 . These E. coli STs have been also observed in humans and livestock in South Korea 9,22,51 . The bla CTX-M-14 -harboring E. coli ST38 and ST69 were reported in vegetables originating from the Dominican Republic 30 , India 30 , and China 52 . The E. coli ST10 strains producing CTX-M-type ESBL were often detected in vegetables 14,44,53 . To the best of our knowledge, this is the first report of ESBL-EC ST354 in vegetables. Furthermore, multiple ESBL-ExPEC clones, including ST131, ST405, ST410, and ST393, have been detected in raw vegetables 28,30 . These results suggest the potential roles of vegetables as the reservoir and propagator for ESBL-ExPEC lineages, which increases the threat of human health as a result of direct consumption.
It has been proposed that ESBL genes, plasmids, and strains may circulate among humans, animals, and the environment 13,54,55 . Notably, livestock functions as a primary reservoir of ESBL producers 17 , and the manure of animal feces contaminated with ESBL-ECs can be recycled as organic fertilizer during vegetable production 56 . In addition, ESBL-ECs have been found abundantly in waterbodies, such as surface water, irrigation water, and wastewater [57][58][59] , which can also be used in agricultural fields. Fecal bacteria can survive for a long time in soil, manure, and water 60 . Thus, fresh vegetables may have acquired resistance through direct or indirect contact with inoculum sources from the contaminated environment.
In conclusion, our findings showed the low prevalence rate of ESBL-ECs in vegetables but the presence of ESBL-ExPECs (ST10, ST38, ST69, and ST354) in stem and leafy, raw vegetables. In particular, the vegetables contaminated with human-associated MDR-ExPEC clones may give rise to further public health concerns and cause epidemics worldwide. Thus, our study emphasizes the importance of intensive monitoring and intervention for antimicrobial resistance from the perspective of the One Health approach, encompassing humans, animals, food produce, and the environment.

Methods
Study design. A total of 1324 raw vegetables, including 879 leafy, 236 fruit, 170 stem, 37 root, and two other types of vegetables, were collected from the Incheon's two largest agricultural wholesale markets (Sam-San and Guwol markets) between February and October 2018. The countries of origin were South Korea (1318 samples), China (4 samples), and New Zealand (2 samples) (Fig. 1). The samples (30-60 g) were homogenized in a tenfold volume of E. coli (EC) broth (Difco Laboratories, Detroit, MI, USA) for 2 min and incubated at 37 °C overnight. Next, 1 ml of enriched media was added to 9 ml of Tryptone Soya Broth (Oxoid, Basingstoke, UK) supplemented with 0.4 g/ml vancomycin (Wako Pure Chemical Industries, Osaka, Japan), followed by incubation at 37 °C for 4 h. The enrichment was streaked on MacConkey agar containing 2 μg/ml cefotaxime. A non-duplicate colony of pink or reddish hue was picked and further grown on CHROMagar ESBL (CHROMagar, Paris, France) to obtain pure cultures. The E. coli species of isolates was determined using matrix-assisted laser desorption ionization-time of flight mass spectrometry (Bruker Daltonik GmbH, Bremen, Germany) with score values ≥ 2.0. To confirm ESBL production, the double-disk synergy test was performed using amoxicillin-clavulanic acid (20/10 μg), cefotaxime (30 μg), ceftazidime (30 μg), and cefepime (30 μg) disks. For the colistin (CST) susceptibility, the minimum inhibitory concentration was determined by the broth microdilution method using the Sensititre system (Thermo Fisher Scientific, Waltham, MA, USA). The phenotypes of resistance, intermediate resistance, and susceptibility were interpreted in accordance with the guidelines of the Clinical and Laboratory Standards Institute document M100-S27 61 , except for tigecycline breakpoints, which were interpreted based on the European Committee on Antimicrobial Susceptibility Testing breakpoint tables version 7.1 62 . Multidrug resistance (MDR) was defined as non-susceptibility to at least one antimicrobial agent of three or more classes 63 . E. coli ATCC 25922 was used as control strain.

Data availability
All data analyzed during this study are included in this published article.