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Vegetables and Fruit as a Reservoir of β-Lactam and Colistin-Resistant Gram-Negative Bacteria: A Review

Antibacterial resistance is one of the 2019 World Health Organization’s top ten threats to public health worldwide. Hence, the emergence of β-lactam and colistin resistance among Gram-negative bacteria has become a serious concern. The reservoirs for such bacteria are increasing not only in hospital settings but in several other sources, including vegetables and fruit. In recent years, fresh produce gained important attention due to its consumption in healthy diets combined with a low energy density. However, since fresh produce is often consumed raw, it may also be a source of foodborne disease and a reservoir for antibiotic resistant Gram-negative bacteria including those producing extended-spectrum β-lactamase, cephalosporinase and carbapenemase enzymes, as well as those harboring the plasmid-mediated colistin resistance (mcr) gene. This review aims to provide an overview of the currently available scientific literature on the presence of extended-spectrum β-lactamases, cephalosporinase, carbapenemase and mcr genes in Gram-negative bacteria in vegetables and fruit with a focus on the possible contamination pathways in fresh produce.


Introduction
Fresh produce is considered a good source of minerals, vitamins, phytonutrients and dietary fiber. Accordingly, there is a consensus that a diet rich in vegetables and fruit may decrease the risk of heart diseases and protect against some types of cancer [1]. In 2003, the Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) started an initiative worldwide to promote fruit and vegetable intake for health, with a recommended minimum consumption of 400 g of vegetables and fruit per day [2]. Following this recommendation, the intake of fresh produce as ingredients in healthy diets has been increasing and has gained popularity globally [3]. Consequently, the consumption of contaminated fresh produce, such as vegetables and fruit eaten raw, has been associated with an increasing number of outbreaks of foodborne disease [1]. In addition, fresh produce represents a route of human exposure to antibiotic-resistant bacteria and has often served as a reservoir of antibiotic resistance genes, representing a major public health threat [3,4].
In this context, one of the main public health preoccupations worldwide is the emergence of Gram-negative bacteria displaying resistance to oxyimino-cephalosporins (3GCs), carbapenem and colistin [3]. β-lactams, essentially extended-spectrum cephalosporins and carbapenems, are the main therapeutic choices to treat infections caused by resistant Gramnegative bacteria [5][6][7]. However, resistance to these antibiotic drugs has been increasing in recent years mostly through β-lactamase production. Various β-lactamases have been identified worldwide, including penicillinases, extended-spectrum β-lactamases (ESBLs), cephalosporinases (AmpC), and carbapenemases [7]. Given these circumstances, the approved alternative is colistin, but its re-use in clinical practice has led to the appearance of colistin-resistant bacteria, particularly through horizontal transfer (mcr) [8].
The transfer of these multidrug-resistant Gram-negative bacteria to fresh produce may occur during production via animal manure, through the use of contaminated irrigation water, or be linked to humans during the post-harvest stage, as well as during transport, conservation and processing by handlers [9]. The ingestion of antibiotic-resistant bacteria poses a potential public health concern since they are able to colonize the gut and exchange resistance genes with intestinal bacteria during their passage through the intestines which facilitates their further dissemination in the environment [3]. Extendedspectrum β-lactamase, cephalosporinase and carbapenemase producers as well as mcr gene-producing Gram-negative bacteria isolated from fresh vegetables and fruit have been reported in several countries around the world [3,4,10,11].
Thus, the aim of this review is to highlight the current situation of the worldwide dissemination of ESBL, cephalosporinase, carbapenemase and mcr gene-producing Gramnegative bacteria from fresh vegetables and fruit, their genetic characteristics, and possible contamination pathways.

β-Lactam Resistance and Gram-Negative Bacteria
β-lactam resistance in Gram-negative bacteria can be attributed to two main mechanisms, these include the acquisition of β-lactamase genes, as well as the modification of the target (penicillin-binding proteins) [12]. β-lactamase enzymes have played an important clinical role and have served as the principal resistance mechanism detected for β-lactam drugs [13,14].
In this worrisome situation, carbapenems were introduced to clinics in the late 1980s and showed significant activity in the treatment of infections caused by AmpC and ESBLproducing Gram-negative bacteria [16,20]. The first carbapenemase reported in Enterobacteriaceae was the Serratia marcescens enzyme (SME-1) in London in 1982. Since then, various carbapenemase enzymes belonging to the Ambler class A β-lactamases have been reported, including imipenemase (IMI-1) and non-metallocarbapanemase class A (NmcA); however, the K. pneumoniae-carbapenemase (KPC) type was the most commonly found [5,16,21]. On the other hand, the first MBL variant was discovered in Bacillus cereus in 1966 and was called the BCII enzyme. Until 1989, only four MBL enzymes had been identified and were all chromosomally encoded, therefore they were deemed clinically negligible. Afterwards, various plasmid-encoding class B carbapenemases were described, such as Imipenem-resistant Pseudomonas-type carbapenemases (IMP),Verona integron-encoded MBL (VIM),and recently, New Delhi MBL (NDM) [22,23].In class D β-lactamases, several variants with relatively weak carbapenemase activity have also been reported as carbapenemase enzymes, including OXA-48, OXA-58, OXA-24/40 and OXA-23 [22]. In Enterobacteriaceae, class D carbapenemases are mainly represented by the OXA-48-like enzymes [24].

Colistin Resistance in Gram-Negative Bacteria
Colistin is a cationic polypeptide antibiotic belonging to the polymyxin family [25]. It was described initially in 1947 in Paenibacillus polymyxa, and it is commonly used in human and veterinary medicines, plant cultivation and animal husbandry [25][26][27]. Although in the 1970s its use was discontinued due to its neuro-and renal toxicity, it was reintroduced in the mid-2000s as a last line therapeutic option for the treatment of extensively drugresistant (XDR) Gram-negative infections, such as those caused by carbapenem-resistant GNB [26,28].
The initial target site of colistin is lipopolysaccharide (LPS), more exactly lipid A, located in the outer membrane, which plays a major role in cell permeability. The electrostatic interaction between the cationic region of colistin, which is from the diamino-butyric acid (Dab) residues, and the negatively charged phosphate groups of lipid A, replace the magnesium and calcium ions previously united with the phosphate group. This destabilizes the lipid A and increases the permeability of the outer membrane, leading to the entry of colistin by a self-promoted uptake mechanism and eventual bacterial death [26,29]. Another antibacterial mechanism is the inhibition of a crucial respiratory enzyme, the type II NADH-quinone oxidoreductase (NDH-2) in the bacterial cell membrane [29]. The increased use of colistin has led to the emergence of colistin-resistant strains worldwide [25]. Colistin resistance is mainly achieved by modification of LPS, and consequently the reduced or absent affinity for colistin. This mechanism, although universal in Gram-negative bacteria, may differ between species. The lipid A of LPS undergoes changes, essentially due to the addition of positively charged residues such as phosphoethanolamine (PEtn) and/or 4-amino-4-deoxy-L-arabinose (L-Ara4N). These molecules decrease the overall negative charge of LPS, leading to a smaller electrostatic interaction with the positive charges of colistin that inhibits cell lysis [26,30].
Previously, the genes responsible for most of these additions were thought to be due to chromosomal mutations in genes of a two-component regulatory system, such as pmrAB, PhoPQ, and mgrB, which are not transferable [30]. In late 2015, Liu et al. described the mobilized colistin resistance (mcr-1) gene in an E. coli isolate recovered from livestock in China [8,31]. MCR-1 confers resistance by modifying the colistin target through the action of phosphoethanolamine transferase, which ensures the transfer of phosphoethanolamine (PEA) onto the glucosamine saccharide of lipid A, contributing as in chromosomal resistance to reduce the net negative charge of lipid A and consequently, colistin binding [32]. After the discovery of the mcr-1 gene, nine other mcr gene types (mcr-2 to mcr-10) were identified. The second mobile colistin resistance gene, mcr-2, was found initially in E. coli strains isolated from pigs and calves in Belgium [33]. The gene mcr-3 was identified in E. coli from pigs in China [34], and mcr-4 was reported in Salmonella enterica serovar Typhimurium strains isolated from pigs in Italy [35]. In 2017, anovel transposonassociated phosphoethanolamine transferase gene (mcr-5) was described in d-tartratefermenting Salmonella enterica subsp. enterica serovar paratyphi B in Germany [36]. In 2018, further variants were described; mcr-6 was identified in Moraxella spp. isolated from pigs in Great Britain [37], while mcr-7 and mcr-8 were described in K. pneumoniae strains isolated from animals (chickens and pigs) in China [38,39]. In 2019, a novel variant mcr-9 was reported in a Salmonella enterica serovar Typhimurium strain isolated from a human in Washington State in 2010 [40] and more recently, Wang et al. reported the detection of an mcr-10 variant in an Enterobacter roggenkampii clinical strain in China [41].

Literature Search Strategy and Data Collection
The dissemination of extended-spectrum β-lactamase, cephalosporinase, carbapenemase and MCR-producing Gram-negative bacteria in fresh produce is a major public health threat, since they are a very suitable pathway for the spread of antibiotic-resistant bacteria from farm to fork. Until June 2021, thirty-three molecular studies have revealed the isolation of Gram-negative bacteria producing β-lactamase and mcr genes on fresh vegetables and fruit. They have been used and are accessible through the PubMed database using the following keywords: "ESBL", "AmpC", "KPC", "VIM", "NDM", "IMP", "OXA-48", "mcr", "carbapenem resistance", "fresh vegetables", "vegetables" and "fruit".

Vegetable and Fruit Isolates with ESBL and Cephalosporinase Genes
A total of nineteen molecular studies reporting the isolation of ESBL-producing Gramnegative bacteria and AmpC genes from vegetables and fruit have been described ( Figure 1, Table 1). The first report of ESBL-producing GNB isolates from vegetables and fruit was reported in 2014 in The Netherlands. These bacteria were reported on six vegetable types that are consumed raw (bunched carrots, blanched celery, endive, chicory, iceberg lettuce and radish), and from iceberg lettuce farms. In that study, the bla FONA-5 gene was detected among Serratia fonticola isolates on iceberg lettuce from a farm. In addition, 35 Rahnella aquatilis strains harboring the bla RAHN gene were identified. Of the 35 isolates, 34 strains were producing the bla RAHN-1 , and only one R. aquatilis strain carried the bla RAHN-2 gene [42]. After this publication, this level of resistance has been reported in Europe, Africa, Asia and America. Like isolates from humans, animals and the environment, the CTX-M family is the most prevalent type of ESBL-producing Enterobacteriaceae found in vegetables. Similarly, in an Italian study carried out on fresh vegetables, the authors refer to the detection of different ESBL enzymes, including CTX-M-15, CTX-M-1, SHV-12 and RAHN-1 in twenty isolates (the bla CTX-M-15 gene in C. freundii, E. coli and Pantoea agglomerans, the bla CTX-M-1 gene in Enterobacter cloacae, the bla SHV-12 in E. coli and bla RAHN-1 in R. aquatilis). Whereas only four isolates displayed AmpC production, among the four strains obtained, two Hafnia alvei isolates carried a bla ACC gene and two E. cloacae harbored a bla DHA-1 gene [43]. A study in The Netherlands investigated the prevalence of third-generation cephalosporin (3GC) resistant Gram-negative bacteria on fresh vegetables. A total of 27 Serratia spp. isolates with an ESBL phenotype harboring a bla FONA variant were obtained, including bla FONA-1 (18.5%), bla FONA-2 (37.0%), bla FONA-3 (7.4%), bla FONA-4 (7.4%), bla FONA-5 (18.5%) and bla FONA-6 (11.1%). The bla SHV-12 gene was detected in one E. coli and two Enterobacter spp. strains; however, one R. aquatilis strain harbored the bla RAHN-1 gene [3]. In Switzerland, two studies reported the detection of bla ESBL genes on vegetable samples. In the first study, the authors evaluated the presence of ESBL-producing Enterobacteriaceae in 68 vegetables imported from the Dominican Republic, India, Thailand and Vietnam via the national airport in Zürich, and 101 samples were purchased in the city of Zürich. In total, 60 ESBL producers were retrieved, including bla CTX-Mand bla SHV -producing E. coli . Moreover, bla CTX-M-15 and bla SHV-2 genes were identified in E. cloacae, E. aerogenes and C. sakazakii, respectively [10]. The second study reported the detection of CTX-M group 2, CTX-M-15 and FONA-2 in Kluyvera ascorbata, E. cloacae and S. fonticola isolates from diced tomato, chopped chives and spinach, respectively [44]. A study from Germany described the isolation of seven ESBL-producing E. coli isolates collected by food safety inspectors during 2011-2013 from markets, producers and supermarkets. Of the seven isolates, two strains were positive for bla CTX-M-14 and two other isolates harbored bla CTX-M-15 genes. However, three remaining strains were positive for bla CTX-M-65 , bla CTX-M-125 and bla CTX-M-2 genes, respectively [45]. In addition, the bla TEM , bla SHV , bla CTX-M and bla DHA genes were also reported in Romania in different Enterobacteriaceae species (S. marcescens, E. cloacae, E. coli, Klebsiella oxytoca and Proteus vulgaris) [46].

Vegetables and Fruit Isolates with Carbapenemase Genes
Only eight reports have revealed the isolation of Gram-negative bacteria producing carbapenemase genes from vegetables and fruit ( Figure 1, Table 2). The first study describing carbapenemase-producing Gram-negative bacteria from fresh vegetable samples was published in 2015. Samples were purchased from different retail shops specializing in Asian food from three different cities in Switzerland, imported from Vietnam, Thailand, and India. In this study, only one Klebsiella variicola strain carrying the blaOXA-181gene was isolated from a coriander sample from Thailand/Vietnam, and the obtained isolate coharbored a quinolone resistant gene (qnrS1). These data suggest that the international production of imported fresh vegetables constitutes a possible reservoir for the spread of carbapenemase-producing Gram-negative bacteria, especially Enterobacteriaceae [57].
In Asia, Usui et al. analyzed 130 samples of fresh vegetables collected from seven supermarkets in Japan, 10 out of the 130 samples contained ESBL-producing Pseudomonas spp. including; P. hunanensis, P. putida, P. parafulva, P. beteli, P. mosselii, P. paralactis and P. arsenicoxydans. These isolates harbored the bla SHV-12 or bla TEM-116 ESBL gene [49]. In China, a nationwide survey investigated the prevalence of ESBL-producing Enterobacteriaceae from retail food, where four isolates were obtained. Three were identified as E. coli and one as C. freundii isolated from retail vegetables, including tomatoes, cucumber and coriander. The C. freundii isolate carried bla CTX-M and bla OXA genes, while two E. coli isolates harbored bla CTX-M and bla SHV genes and one other E. coli strain carried bla CTX-M , bla SHV and bla TEM genes [7]. In Malaysia, ESBL or AmpC genes were detected in two E. coli (bla CTX-M-55 and bla CTX-M-65 ) and two K. pneumoniae isolates (bla CTX-M-15 , bla SHV-28 and bla DHA-1 ) from coriander and chili pepper respectively [50]. In addition, different CTX-M variants were described from E. On the American continent, different Enterobacteriaceae isolates harbored bla SHV , bla TEM and bla CTX-M-1 as well as bla CTX-M and bla CMY genes and were detected from iceberg lettuce and leafy greens, respectively in the United States [52,53]. Moreover, seven E. coli isolates carrying the bla CTX-M-15 gene were reported from leaf lettuce, alfalfa and parsley/cilantro in Ecuador [54], while bla CTX-M-14 , bla CTX-M-15 , bla CTX-M-27 , bla SHV-106 and bla SHV-142 -positive Enterobacteriaceae were reported in Canada from imported vegetable samples [55], and the bla CTX-M-15 gene in Brazil [56].

Vegetables and Fruit Isolates with Carbapenemase Genes
Only eight reports have revealed the isolation of Gram-negative bacteria producing carbapenemase genes from vegetables and fruit ( Figure 1, Table 2). The first study describing carbapenemase-producing Gram-negative bacteria from fresh vegetable samples was published in 2015. Samples were purchased from different retail shops specializing in Asian food from three different cities in Switzerland, imported from Vietnam, Thailand, and India. In this study, only one Klebsiella variicola strain carrying the bla OXA-181 gene was isolated from a coriander sample from Thailand/Vietnam, and the obtained isolate coharbored a quinolone resistant gene (qnrS1). These data suggest that the international production of imported fresh vegetables constitutes a possible reservoir for the spread of carbapenemase-producing Gram-negative bacteria, especially Enterobacteriaceae [57].
Since its first report, carbapenemase genes have been identified in bacteria from different vegetable samples in only five countries across the Asian, African and European continents. Indeed, OXA-72-producing Acinetobacter calcoaceticus strains have been found in two vegetable samples purchased from the same market in Beirut, Lebanon [58]. In Japan, two K. pneumoniae and one Acinetobacter baumannii isolate were collected from vegetable samples in the city of Higashi-Hiroshima. Both K. pneumoniae isolates carried bla NDM-1 with other genes conferring resistance to β-lactams (bla CTX-M-15 , bla OXA-9 , and bla TEM-1A ), aminoglycosides (aac(6')-Ib, aadA1, and aph(3')-VI), quinolones (qnrS1) and fluoroquinolones (aac(6')-Ib-cr). While the obtained A. baumannii isolate coharbored bla OXA-66 , bla OXA-72 and genes conferring resistance to sulfonamides (sul2), tetracycline (tet(B)), and streptomycin (strAB) [59]. In China, Wang et al., identified an Escherichia coli strain coproducing bla KPC-2 and bla NDM-1 genes in fresh lettuce from a market in Guangzhou. In addition, this multidrug resistant E. coli strain coharbored fosfomycin resistance genes (fosA3 and floR). This study represents the first report of either bla KPC or bla NDM genes in bacteria obtained from vegetables [60]. Additionally, two other studies from China reported the detection of carbapenemase-positive enterobacterial isolates. The first reported the isolation of twelve carbapenem-resistant isolates obtained from vegetable samples, where the highest detection rate was found in curly endive samples. The authors identified two K. pneumoniae isolates carrying the bla KPC-2 gene, while five of each E. coli and C. freundii strains harbored the bla NDM gene, including four E. coli with the bla NDM-5 gene and five C. freundii with bla NDM-1 . Notably, one E. coli strain from a cucumber sample harbored bla NDM-5 and bla KPC-2 genes simultaneously. All C. freundii and E. coli isolates carried fosfomycin resistance genes (fosA3and floR), and all K. pneumoniae and C. freundii isolates harbored the floR gene. However, one strain of E. coli and C. freundii harbored the aminoglycoside resistance gene (rmtB). Quinolone resistance genes including oqxAB and qnrB, were found in four and eight isolates, respectively [61]. The second report signaled the detection of two E. coli isolates carrying bla NDM genes in leaf rape and spinach recovered from two supermarkets in Shandong province; one isolate concomitantly harbored bla NDM-9 , mcr-1 and fosA3, while the second isolate carried bla NDM-5 , mcr-1 and fosA3 genes [4].
From the African continent only one study from Algeria has been reported. The authors identified three K. pneumoniae isolates harboring the bla OXA-48 gene from lettuce, tomatoes and parsley in Béjaïa city [11]. In Europe and more precisely from Romania, the bla OXA-48 and bla KPC genes were detected in E. cloacae and K. oxytoca isolates from parsley samples [46].

Vegetables and Fruit Isolates with the mcr Gene
To date, eight studies have reported mcr-producing Gram-negative bacteria, especially isolates of Enterobacteriaceae species, from fresh produce that mostly originated from China ( Figure 1, Table 2). The mcr-1 gene was first reported in 2016 in Switzerland in two out of sixty isolates. The two E. coli isolates carried the mcr-1 gene and coharbored bla CTX-M-55 and bla CTX-M-65 genes, respectively [67]. After this first description in fresh produce, mcr-1-producing GNB isolates on fresh produce were reported in China, where seven E. coli and two Raoultella ornithinolytica isolates were recovered from tomato and lettuce samples between May 2015 and August 2016 in Guangzhou. All the obtained mcr-1-positive strains harbored the florfenicol resistance gene (floR). Of the nine isolates, six strains carried the bla CTX-M gene (four bla CTX-M-14 and two bla CTX-M-55 ), with five and four strains harboring the fosA3 and oqxAB efflux pump gene, respectively [64]. Moreover, the mcr-1 gene was described in China from a total of 528 fresh vegetable samples, including 18 different types purchased from 53 supermarkets and farmers markets from 23 districts or cities in nine provinces between May 2017 and April 2018. Of the 528 samples analyzed, only 19 samples harbored one or more mcr-positive isolates, and the three highest detection rates were noted in carrots (14.3%), pakchoi (13.3%) and green pepper (7.7%), followed by leaf lettuce (5.6%), leaf rape (4.9%), romaine lettuce (4.3%), tomato (3.5%), spinach (3.2%), cucumber (3.1%), and curly endive (2.4%). In the above study, twenty-four mcr-1-positive isolates were obtained; twenty-three strains were identified as E. coli and one as E. cloacae. Fourteen mcr-1-positive strains coproduced the bla CTX-M gene, nine strains harbored the bla CTX-M-9G gene and three strains carried bla CTX-M-1G . However, the remaining two strains harbored both bla CTX-M-9G and bla CTX-M-1G genes. In addition, eight and two isolates harbored fosA3 and rmtB genes, respectively. Plasmid-mediated resistance to quinolones (PMQR), including oqxAB, qnrS and qnrB genes, were also detected in this study [66]. Additionally, in the same country, two E. coli isolates carrying the mcr-1 gene were isolated from leaf rape and spinach in Shandong province. These isolates coharbored metalloβ-lactamase and fosA3 genes; the first carried bla NDM-5 , while the second harbored the bla NDM-9 gene [4]. In 2018, one E. coli isolate carrying the mcr-1 gene recovered from lettuce was reported in South Korea. The obtained isolate coharbored bla TEM-1 and bla CTX-M-55 genes [65]. In Portugal, the mcr-1 gene was reported by Manageiro et al. in 2020, and they documented the presence of this gene in an E. coli strain isolated from a lettuce sample. In silico analysis showed the presence of additional antibiotic resistance genes including bla TEM-1 , aac(3)-Iv, aadA1, aph(4)-Ia, aph(6)-Id, mdf(A)-type, tetA, sul2 and floR-type [63]. Another report from Portugal revealed the detection of the mcr-1 gene in an E. coli strain from conventionally produced lettuce. The isolate co-carried bla TEM-1 , aph(4)-Ia, floR, sat2, strA, strB, sul2 and tetA genes, while no conventional and organic fruit were positive for the mcr-1 gene [62].
On fruit samples, the mcr-1 gene was detected in E. coli and K. pneumoniae isolates from apple and orange samples recovered in China [68].

Contamination Pathways and Genetic Characteristics of β β β-Lactamases and mcr-Producing Gram-Negative Bacteria
The high diversity of global clones illustrates the extensive spread of ESBL-producing K. pneumoniae and E. coli isolates on vegetables around the world (ST45, ST219, ST15 and ST147 found in K. pneumoniae isolates, and ST410-A, ST44, ST405, ST131 and ST38 in E. coli isolates). In Algeria, sequence type 14, ST45, ST219, ST236, and ST882 have been identified among K. pneumoniae strains recovered on fresh fruit and vegetables carrying ESBL or cephalosporinase genes, including bla CTX-M-15 , bla OXA-1 , bla SHV-101 , bla SHV-28 and bla DHA-1 genes that were mostly (11 of 13) located on the IncFII plasmid, while the IncR plasmid replicon was identified in only one isolate [48]. In Switzerland, twenty-two different sequence types identified in E. coli-positive ESBL have been described on imported vegetables, including ST4684 and ST4683, four of them belonging to the epidemiologically important sequence types ST405 (n = 1), ST131 (n = 2) and ST38 (n = 2) [10]. Similarly, the ST131 E. coli clone is known for its role in the global spread of ESBLs, especially CTX-M-15, and this clone has had an inevitable clinical impact on antibiotic resistance and pathogenicity [69]. In the same study conducted in Switzerland, high clonal diversity was observed among K. pneumoniae strains, with two isolates belonging to the epidemic clones ST15 and ST147 [10]. In Quito, Ecuador, the hyper epidemic clones ST410 and ST44 harboring the bla CTX-M-15 gene have been identified in E. coli isolates from leaf lettuce, alfalfa and parsley/cilantro, and three of them were found on the same integron 1 variable region (dfrA1 and addA5). The five remaining isolates presented bla CTX-M-15 downstream of an insertion sequence element p1 (ISEcp1) [54]. In a survey in Germany, a high diversity of global clones was identified in ESBL-producing E. coli isolates from different vegetable samples. These ESBL determinants were detected on different plasmids as follows: IncHI2 and IncK (bla CTX-M-14 , ST527, ST973), IncN (bla CTX-M-65 , ST10), IncFIB (bla CTX-M-15 , ST410), IncHI2 (bla CTX-M-125 , ST542) and IncFIA-FIB (bla CTX-M-2 , ST120) [45]. The IncFIB and IncFIC plasmid replicons were found in Pseudomonas hunanensis, and P. putida carried the bla SHV-12 respectively.The plasmid IncK/B was reported in P. paralactis harboring the bla TEM-16 gene [49].
The dissemination of carbapenemase-producing E. coli is polyclonal, where multiple STs have been reported. The sequence type 877 was reported in an E. coli strain coproducing bla KPC-2 and bla NDM-1 genes isolated from fresh lettuce. These genes were located on 64 and 118 kb plasmids, designated as plasmids pHNTS79-KPC and pHNTS79-NDM, respectively [60]. Furthermore, ST1877 and ST391 have been detected in OXA-48-positive K. pneumoniae isolates from lettuce, tomatoes and parsley in Algeria, where the ST391 clone is considered as an emergent carbapenemase-producing lineage of clinical importance [11]. The bla OXA-181 detected in a Klebsiella variicola isolate from a coriander sample was mediated by the IncX3-type plasmid of 51-kb [57]. Indeed, in Japan the epidemic clones ST15 and ST2 were reported among K. pneumoniae that carried the bla NDM-1 gene and the A. baumannii strain coharbored bla OXA-66 , and bla OXA-72 genes, respectively [59]. ST15 is a relatively common NDM-positive K. pneumoniae lineage, and it has been found in various countries across different continents, almost all of which were isolated from humans [70]. IncX3 plasmids carrying the bla NDM gene have been identified in E. coli and C. freundii strains isolated from cucumbers, and the identified IncX3 plasmid was identical or highly similar (99%) to the IncX3 plasmids identified from patients in other countries. In addition, similar F35:A-:B1 plasmids were described in two bla KPC-2 -producing-K. pneumoniae isolates belonging to ST23 obtained from different cities. Two E. coli isolates carrying the bla NDM-5 gene isolated from cucumber and romaine lettuce samples in different cities in China shared an identical PFGE pattern and sequence type (ST4762); however, one E. coli strain belonged to ST167 [61].
Among E. coli isolates, the mcr-1 gene was found in multiple STs from different countries, ST10 in South Korea, and ST167 and ST4683 in Switzerland [65,67]. ST156 and ST2847 have been identified in China from leaf rape and spinach samples. In the latter, mcr-1 genes were located on the~60-kb IncI2 plasmid or the~33-kb IncX4 plasmid; even as bla NDM-5 was on the~46-kb IncX3 plasmid while bla NDM-9 was on the~120-kb untypeable plasmid. The detected plasmids were highly similar to those from patients and animals described in different countries [4]. In a Chinese study, six sequence types, including ST795, ST2505, ST69, ST156, ST48 and ST206, were described in seven E. coli isolates recovered from lettuce and tomato samples. For the four E. coli isolates, mcr-1 genes were located on IncHI2, IncI2 or IncX4 plasmids, while for the two Raoultella ornithinolytica isolates, the mcr-1 gene was located on the IncHI2 plasmid [64]. Moreover, in China, sixteen STs along with a new ST type were identified, while the most prevalent STs were ST744 and ST224. In this study, different plasmid replicons were detected, including IncX4, IncI2 and IncHI2; where IncX4 was the most detected and shared highly similar RFLP profiles, although they were from different cities and fresh vegetables. The mcr-1 gene was located on the IncX4 plasmid of ∼33 kb in size [66].
In addition to the above results, the study found that patients and animals shared identical or highly similar plasmids with vegetables [4,61]. Various other studies reported that vegetables may become contaminated with multidrug resistant bacteria from soil, manure fertilization, irrigation water or through direct contamination by humans [2]. In this context, the major way in which antibiotic resistance enters the soil is through the use of animal manure [71]. In Australia, Zhang et al. explored the impact of cattle and poultry manure application on the resistome in lettuce and soil microbiomes, including the rhizosphere, root endosphere, leaf endosphere and phyllosphere. In addition, they identified potential transmission routes of antibiotic resistance genes in the soil-plant system. The authors reported that poultry manure application increased antibiotic resistance genes in the rhizosphere, root endophyte and phyllosphere, while cattle manure use increased the abundance of antibiotic resistance genes only in the root endophyte, suggesting that poultry manure may have a stronger impact on lettuceresistomes. Moreover, the authors also identified an overlap of antibiotic resistance gene (ARG) profiles between lettuce tissues and soil, which indicates that plant and environmental resistomes are interconnected, and confirmed the transmission of antibiotic resistance genes from manured soil to vegetables. Two main transmission pathways were reported: an internal pathway through plant tissues and an external pathway via aerosol from the atmosphere to the plant surface. Thus, in the external pathway, sixty-nine ARGs were shared between poultry manure-amended soil and the phyllosphere of lettuce, while in the internal pathway 47 genes were common between rhizosphere soil and the root endophyte [72]. This finding was consistent with previous studies which reported that the phyllosphere resistome was significantly more abundant and diverse than the endophytic resistome in organic vegetables [73,74]. Another report from China described the impact of the long-term use of inorganic (chemical) and organic (manure) fertilizers on antibiotic resistance genes in greenhouse soils growing vegetables. The results showed that both inorganic and organic use increased the abundance and the diversity of soil ARGs, with a difference in the dominant ARG types. ARG abundance and diversity were both higher in organic fertilizer [75]. These data confirmed those of previous reports indicating that fertilizer application, especially organic fertilizer such as animal manure, raised ARG abundance and diversity in soil compared to soil without fertilization [75][76][77].
Several studies have reported food-borne human outbreaks linked to the consumption of fresh vegetables and fruit irrigated with wastewater and indicated that the type of irrigation practice plays a vital role in the contamination of farm produce [78]. In this context, Araújo et al. characterized the presence of E. coli isolates on vegetables and in irrigation water sampled from 16 household farms in Portugal. In this later study, different commonly acquired genes such as bla TEM , tetA and tetB and plasmids (IncFIC, IncFrep and IncFIB) were detected in isolates in both water and vegetable samples. In addition, rep-PCR typing results detected the same STs and identical clones in vegetables and water, suggesting cross-contamination. These results suggest that irrigation groundwater is a reservoir of antibiotic resistant E. coli and may enter the food chain via vegetable consumption [79]. Makkaew [80]. In Ghana, Antwi-Agyei et al. reported that irrigation with partially treated and untreated wastewater is a key risk factor for the observed contamination of 80% of produce samples, with a median concentration ranging from 0.64 to 3.84 log E. coli/g produce, while ready-to-eat salad was the most contaminated with 4.23 log E. coli/g [81].

Conclusions
This review provides a reference for an enhanced understanding of the global risk of fresh vegetables and fruit in the transmission of multidrug resistant Gram-negative bacteria and emphasizes the necessity of paying close attention to these products as a future public health issue. Given that fresh produce is often consumed raw, this allows the transfer of these antibiotic resistance genes to human gut bacteria. It is now even more important that more investigations should be performed in order to survey the emergence and transmission of these genes to humans from farm to fork. In addition, suitable measures, including the improvement of water quality and agricultural practices, need to be considered to ensure consumer safety worldwide.