The transferable resistome of produce

Abstract

25Produce is increasingly recognized as a reservoir of human pathogens and transferable 26 antibiotic resistance genes. This study aimed to explore methods to characterize the 27 transferable resistome of bacteria associated with produce. Mixed salad, arugula, and 28 cilantro purchased from supermarkets were analyzed by means of cultivation-and DNA-29 based methods. Before and after a nonselective enrichment step, tetracycline (tet) 30 resistant Escherichia coli were isolated and plasmids conferring tet resistance were 31 captured by exogenous plasmid isolation. Tet resistant E. coli isolates, transconjugants 32 and total community (TC)-DNA from the microbial fraction detached from leaves or after 33 enrichment were analyzed for the presence of resistance genes, class 1 integrons and 34 various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time 35 PCR primers were developed for IncI and IncF plasmids. Tet resistant E. coli isolated 36 from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU and IncX1 plasmids. 37 Three isolates from cilantro were positive for IncN plasmids and bla CTX-M-1 . From mixed 38 salad and cilantro, IncF, IncI1, and IncP-1β plasmids were captured exogenously. 39 Importantly, whereas direct detection of IncI and IncF plasmids in TC-DNA failed, these 40 plasmids became detectable in DNA extracted from enrichment cultures. This confirms 41 that cultivation-independent DNA-based methods are not always sufficiently sensitive to 42 detect the transferable resistome in the rare microbiome. In summary, this study 43showed that an impressive diversity of self-transmissible multiple resistance plasmids 44 was detected in bacteria associated with produce that is consumed raw, and exogenous 45 capturing into E. coli suggests that they could transfer to gut bacteria as well. 46 47 48Produce is one of the most popular food commodities. Unfortunately, leafy greens can 49 be a reservoir of transferable antibiotic resistance genes. We found that IncF and IncI 50 plasmids were the most prevalent plasmid types in E. coli isolates from produce. This 51 study highlights the importance of the rare microbiome associated with produce as a 52 source of antibiotic resistance genes that might escape cultivation-independent 53 detection, yet may be transferred to human pathogens or commensals.

Exploring the Animal Waste Resistome: The Spread of Antimicrobial Resistance Genes Through the Use of Livestock Manure

“…IncP-1, a common environmental plasmid group, is largely known for its efficient conjugative transferability potential and stable replication in a wide range of Gram-negative bacteria (Heuer et al, 2012). Conversely, plasmids IncF (Villa et al, 2010), IncI (Blau et al, 2018), and IncQ (Rawlings and Tietze, 2001) show a narrower host range. These plasmids are assumed to be important for the dissemination of ARG in Escherichia coli and other Enterobacteriaceae (Johnson and Nolan, 2009;Suzuki et al, 2010;Heuer et al, 2012;Van Houdt et al, 2013).…”

“…Exogenous plasmid isolation techniques (Bale et al, 1988) clarified how plasmids diffuse in different environments. Recently, plasmids from municipal sewage sludge and recipient bacteria were analyzed for their transferability by exogenous isolation (Blau et al, 2018;Wolters et al, 2018). Referring to pig manure samples, four IncQ-like plasmids were isolated in recipient strains: Pseudomonas putida UWC1, Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and E. coli.…”

“…The acquisition of AMR by bacteria may be due to spontaneous mutations (Woodford and Ellington, 2007) or, more frequently, by gaining specific ARGs from other bacteria through HGT. High density of microbial cells in the presence of antimicrobial compounds and nutrients, as observable in manure (Blau et al, 2018), triggers HGT events among bacteria, thus conferring selective advantage to the hosts (Thomas and Nielsen, 2005). Mutations are essential for the continuous evolution of ARGs, producing hundreds of variants which are hardly identifiable and increasingly dangerous for the environment (Woodford and Ellington, 2007).…”

Persistence of wastewater antibiotic resistant bacteria and their genes in human fecal material

“…The blaTEM gene may have been ingested by the donor (e.g. fresh produce) (Blau et al, 2018), and its occurrence in the gut microbiota of healthy individuals, even if never exposed to antibiotics, has been reported (Fouhy et al, 2014;Sommer et al, 2009). In general, the vanA gene abundance (per gram of dry weight) did not vary significantly from T0 to T7, with average measurements at T0 and T7 of 5.87 ± 0.11 and 5.79 ± 0.15 log-units under aerobic conditions, and 7.34 ± 1.17 and 7.17 ± 1.12 log-units under anaerobic conditions (Figure 2).…”

“…Indeed, several recent studies have shown the presence of ARB and ARGs in raw-eaten products (e.g. lettuce), raising concerns for consumers, particularly for immunocompromised people (Araujo et al, 2017;Blau et al, 2018;O'Flaherty et al, 2019;Zhang et al, 2017;Zhu et al, 2017). However, even after ingestion, the success of allochthonous bacteria in the intestinal tract is supposedly antagonized by the gut microbiota, a complex and dynamic community of microorganisms colonizing the gastrointestinal tract of humans since birth (Donaldson et al, 2016;Gibson et al, 2014).…”