Produce is one of the most popular food commodities. Unfortunately, leafy greens can be a reservoir of transferable antibiotic resistance genes. We found that IncF and IncI plasmids were the most prevalent plasmid types in E. coli isolates from produce. This study highlights the importance of the rare microbiome associated with produce as a source of antibiotic resistance genes that might escape cultivation-independent detection, yet may be transferred to human pathogens or commensals.

The persistence of Salmonella in the environment is influenced by a multitude of biotic and abiotic factors. In addition, its persistence can be influenced by preadaptation before the introduction into the environment. In order to study how preadaptation changes the survival of Salmonella in soil and therefore its potential to colonize the phytosphere, we developed a new medium based on lettuce material [lettuce medium (LM)]. Salmonella enterica serovar Typhimurium strain LT2 was used as a model for Salmonella in this study. LT2 was inoculated into soil microcosms after pregrowth in Luria Bertani (LB) broth or in LM. Survival of LT2 in soil was monitored over 56 days by plate counts and quantification of the Typhimurium-specific gene STM4497 using qPCR in total community DNA for which primers and TaqMan probe were designed in this study. Significantly enhanced persistence was observed for LT2 pregrown in LM compared to LT2 pregrown in LB, indicating a preadaptation effect. Surprisingly, no improved survival could be observed for S. Typhimurium strain 14028s and S. enterica serovar Senftenberg after pregrowth on LM. This indicates a high strain specificity of preadaptation. Results from previous studies suggested that biofilm formation could enhance the survival of human pathogens in various environments and might contribute to enhanced survival on plants. In vitro biofilm assays with several Salmonella strains revealed a strain-specific effect of LM on the biofilm formation. While LM significantly improved the biofilm formation of S. Senftenberg, the biofilm formation of LT2 was better in LB. This indicates that the better survival of LM-pregrown LT2 in soil was not linked to an improved ability to form biofilms but was likely due to other factors. Most importantly, this study showed that the medium used to pregrow Salmonella can influence its survival in soil and its biofilm formation which might influence the fate of Salmonella in soil.

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.

No abstract

Fruits and vegetables are important for a healthy diet. However, when eaten raw and contaminated with human pathogens (HPs) they may cause a disease outbreak. Contamination with HPs can occur along the entire farm-to-fork production chain and Salmonella enterica is one of the most common foodborne pathogens. A range of biotic and abiotic environmental factors can inluence the complex interactions between Salmonella and plants. Moreover, the outcome of experiments largely depends on the experimental design and parameters or methods employed, and on top, on the accompanying plant microbiome and the genetic equipment of the plant and the Salmonella strain. Particularly mobile genetic elements contribute to the diversiication and adaptation of Salmonella to the plant environment. So far, litle is known about the key processes and factors inluencing the atachment and potential internalization of Salmonella in plants and the plant speciic responses. It is therefore important to beter understand the ecology of Salmonella in the soil and plant environment, in order to propose practicable recommendations for prevention of foodborne diseases. This also requires improved sensitivity and speciicity of detection methods. In this chapter, we present the current knowledge, research needs, and methodology regarding the complex interactions between Salmonella and plants.