Rhizosphere effect on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure-amended soil during cabbage (Brassica oleracea) cultivation under tropical field conditions in Sub-Saharan Africa
Research highlights
► Rhizosphere effect depends on inoculum density. ► No rhizosphere effect at 4 log CFU/g. ► Rhizosphere enhances persistence in manure-amended soil at 7 log CFU/g. ► Enhanced persistence is associated with long-term contamination of the leaves.
Introduction
Microbiological safety concerns attributed to the contamination of fresh produce with potential human pathogens such as E. coli O157:H7 and Salmonella spp. is well recognised (Barak et al., 2005, Doyle and Erickson, 2008, Nguyen-the and Carlin, 2000). Earlier studies aimed at finding solutions for microbiological safety problems of fresh vegetables suggest that sanitation of fresh produce can be executed at post-harvest level. Sanitation methods used are mostly based on a chemical decontamination step expected to ensure microbiological safety without loss of produce quality (Gopal et al., 2010, López-Gálvez et al., 2010, Vandekinderen et al., 2009, Weissinger et al., 2000). However, frequent outbreaks of E. coli O157:H7, Salmonella spp. and other microbial infections associated with fresh-cut vegetables raised concerns about the efficacy of such post-harvest sanitation approaches in guaranteeing the safety of fresh produce (Ölmez and Kretzschmar, 2009). Internalisation of human pathogenic microorganisms in plant tissues is probably one of the factors that limit the efficacy of sanitizers since such cells are completely protected from the biocides (Dong et al., 2003, Itoh et al., 1998, Solomon et al., 2002, Takeuchi and Frank, 2000, Takeuchi and Frank, 2001). Moreover, currently consumers have a preference for food that is free of additives. This shift in consumer behaviour discourages the use of post-harvest sanitizers for fresh produce decontamination. In addition, some chemical sanitation agents can result in the generation of potential hazardous by-products (Gil et al., 2009). For example, the reaction of chlorine with natural organic matter has been shown to generate by-products such as chloroform, haloacetic acids and trihalomethanes, which can elicit carcinogenic or mutagenic effects (Hua and Reckhow, 2007, Nieuwenhuijsen et al., 2000).
The inadequacy of post-harvest sanitizers to inactivate pathogenic organisms on fresh produce coupled with potential human health risks associated with in-situ-generated by-products strongly underscores the need to produce safe raw materials at pre-harvest level. Design of strategies to control pre-harvest vegetable contamination in the field requires thorough understanding of the factors that affect the survival of potential pathogens in the soil–plant ecosystem during cultivation. One such factor is the rhizosphere. The rhizosphere is a nutrient-rich ecological niche where the soil is under direct influence of the plant roots and where competition within the microbial community has been shown to be fiercely high (Berg et al., 2002). Since soil is not the primary habitat of E. coli O157:H7 and Salmonella spp., the rhizosphere of cultivated vegetable crops could either enhance their survival in soil increasing the risk of contamination of edible plant parts at harvest or reduce their survival and thus prevent contamination. Previous research conducted with cereals to elucidate the effect of the rhizosphere on survival of enteric pathogenic bacteria provided contradictory results. Gagliardi and Karns (2002) demonstrated enhanced persistence of E. coli O157:H7 in soils in the rhizosphere of maize compared to bulk soil. Williams et al. (2007) showed that the survival of E. coli O157:H7 in the rhizosphere of maize grown in waste-amended soil was affected by waste type but unaffected by the rhizosphere. Recently, Semenov et al. (2009) reported a significant positive effect of the rhizosphere of lettuce plants on average cell density of E. coli O157:H7 and S. Typhimurium in soil under controlled green house conditions (16 °C and 50% RH). However, we are not aware of any previous reports on the effect of rhizosphere of vegetable plants on survival of enteric food-borne pathogens in manure-amended soil under tropical field conditions as found in Sub-Saharan Africa. In previous research, we observed that E. coli O157:H7 and S. Typhimurium appear to survive longer in manure-amended soil on which cabbages were cultivated (Ongeng et al., 2011a) than in manure-amended bulk soil (Ongeng et al., 2011b) under field conditions in the Central Agro-Ecological Zone (CAEZ) of Uganda (Wortman and Eledu, 1999). The longer survival observed in the presence of plant roots suggested the possibility of a rhizosphere effect. However, no conclusions could be drawn about the rhizosphere effect since survival in bulk soil and in cultivated soil were determined in different experiments. The objective of this study was to determine whether under tropical field conditions encountered in the CAEZ of Uganda, the rhizosphere of cabbage plants influences survival of E. coli O157:H7 and S. Typhimurium in manure-amended soil, and to assess transfer of the pathogens to the foliage during cultivation.
Section snippets
Bacterial strains and culture media
Rifampicin-resistant derivatives of non-virulent E. coli O157:H7 ATCC 43888 (E. coli O156:H7-Rifr) and S. Typhimurium LT2A (S. Typhimurium-Rifr) were used. Escherichia coli O157:H7 ATCC 43888 lacks the genes for shiga-like toxins 1 (stx1) and 2 (stx2). The stx1 and stx2 genes in E. coli O157:H7 did not influence bacterial survival in bovine manure and manure slurry (Kudva et al., 1998). S. Typhimurium LT2A is a virulence-attenuated laboratory strain due to a mutation in the rpoS gene. The
Survival of E. coli O157:H7-Rifr in cabbage rhizosphere and in bulk soil
In the case of an initial inoculum density of 4 log CFU/g, CFU of E. coli O157:H7-Rifr in the rhizosphere of cabbage plants and in bulk soil declined till below the detection limit of the plate count method at 21 and 24 days post-transplantation, respectively (Fig. 2A). At each sampling point, CFU number was not significantly different between the rhizosphere and bulk soil (p > 0.05). Enrichment showed that the organism remained at low numbers in the rhizosphere and in bulk soil till day 27.
Discussion
In this study, we examined whether under tropical field conditions, the rhizosphere of cabbage plants influences survival of E. coli O157:H7 and S. Typhimurium in manure-amended soil. Therefore, we compared persistence times of E. coli O157:H7-Rifr and S. Typhimurium-Rifr in the rhizosphere of cabbage plants with those in bulk soil. The results revealed that, both for E. coli O157:H7-Rifr and S. Typhimurium-Rifr, the 7 log CFU/g inocula survived longer in the rhizosphere than in bulk soil
Conclusion
This study demonstrated that the effect of rhizosphere of cabbage plants on survival of E. coli O157:H7 and S. Typhimurium in manure-amended soil under tropical field conditions in Sub-Saharan Africa depended on inoculum density. The study indicated clearly that: i) the rhizosphere of cabbage plants enhanced the persistence of the 7 log CFU/g E. coli O157:H7-Rifr and S. Typhimurium-Rifr but had no effect on survival of the 4 log CFU/g inoculum; and ii) enhanced persistence of the 7 log CFU/g E.
Acknowledgements
This research was sponsored by the International Foundation for Science (Ref. C/4294-1) and the Belgian Development Agency (UNI2006/01).
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