Sewage sludge amendment and inoculation with plant-parasitic nematodes do not facilitate the internalization of Salmonella Typhimurium LT2 in lettuce plants Eva Fornefeld a , Mohamed Baklawa a, b , Johannes Hallmann c , Adam Schikora a , Kornelia Smalla a, * a Julius Kühn-Institut, Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany b Suez Canal University, Faculty of Agriculture, Agricultural Botany Department, Ismailia, Egypt c Julius Kühn-Institut, Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Münster, Germany article info Article history: Received 1 November 2016 Received in revised form 13 June 2017 Accepted 13 June 2017 Available online xxx Keywords: Salmonella enterica Typhimurium LT2 Sewage sludge Meloidogyne hapla Pratylenchus crenatus Internalization Soil Survival Lettuce abstract Contamination of fruits and vegetables with Salmonella is a serious threat to human health. In order to prevent possible contaminations of fresh produce it is necessary to identify the contributing ecological factors. In this study we investigated whether the addition of sewage sludge or the presence of plant- parasitic nematodes foster the internalization of Salmonella enterica serovar Typhimurium LT2 into let- tuce plants, posing a potential threat for human health. Greenhouse experiments were conducted to investigate whether the amendment of sewage sludge to soil or the presence of plant-parasitic nema- todes Meloidogyne hapla or Pratylenchus crenatus promote the internalization of S. Typhimurium LT2 from soil into the edible part of lettuce plants. Unexpectedly, numbers of cultivable S. Typhimurium LT2 decreased faster in soil with sewage sludge than in control soil but not in root samples. Denaturing gradient gel electrophoresis analysis revealed shifts of the soil bacterial communities in response to sewage sludge amendment and time. Infection and proliferation of nematodes inside plant roots were observed but did not influence the number of cultivable S. Typhimurium LT2 in the root samples or in soil. S. Typhimurium LT2 was not detected in the leaf samples 21 and 49 days after inoculation. The results indicate that addition of sewage sludge, M. hapla or P. crenatus to soil inoculated with S. Typhi- murium LT2 did not result in an improved survival in soil or internalization of lettuce plants. © 2017 Published by Elsevier Ltd. 1. Introduction Contamination of fruits and vegetables with bacterial human pathogens like Salmonella enterica or Escherichia coli O157:H7 is a serious concern (Brandl, 2006). Internalized bacteria cannot be removed by washing or disinfection and can pose a risk for human health when contaminated produce is eaten uncooked. Increasing cases of human illnesses associated with raw fruits and vegetables have raised interest in the identification of sources of contamina- tion and ecological factors determining the fate of human patho- gens in the environment (Wiedemann et al., 2015). Contamination of plants can occur along the whole farm-to-fork production chain. One possible source of contamination is field soil contaminated with human pathogens (Maffei et al., 2017; Ongeng et al., 2014, 2015; Silva et al., 2014). Soil can be infected by various routes, e.g. by fecal contaminations, contaminated water or fertilizer. Sewage sludge is routinely utilized on agricultural land to increase organic matter content and improve nutrient availability in soil (Casado- Vela et al., 2007; Gasco and Lobo, 2007). However, there are po- tential risks when using sewage sludge as fertilizer. Sewage sludge often contains heavy metals or bacteria carrying resistance genes, integrons and mobile genetic elements such as plasmids or con- jugative transponsons (Calero-Caceres et al., 2014; Tennstedt et al., 2003), as well as human pathogens (Singh and Agrawal, 2008). Since the majority of human pathogenic bacteria are not able to degrade plant tissues they rely on natural openings or wounds for internalization into plants (Teplitski et al., 2009; Tyler and Triplett, 2008). Plant-parasitic nematodes can provide these entry sites and in addition serve as a vector transporting bacteria towards plants * Corresponding author. Julius Kühn-Institut, Institute for Epidemiology and Pathogen Diagnostics, Messeweg 11-12, 38104 Braunschweig, Germany. E-mail address: kornelia.smalla@julius-kuehn.de (K. Smalla). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm http://dx.doi.org/10.1016/j.fm.2017.06.011 0740-0020/© 2017 Published by Elsevier Ltd. Food Microbiology xxx (2017) 1e9 Please cite this article in press as: Fornefeld, E., et al., Sewage sludge amendment and inoculation with plant-parasitic nematodes do not facilitate the internalization of Salmonella Typhimurium LT2 in lettuce plants, Food Microbiology (2017), http://dx.doi.org/10.1016/ j.fm.2017.06.011