Salmonella enterica Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves.

Jeanine Montano,Michael Mcclelland,Joseph Torreano,Steffen Porwollik,Maeli Melotto,Shlomo Sela Saldinger,Gabrielle Rossidivito

doi:10.3389/fmicb.2020.00006

Abstract

Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety.

Highlights

Human pathogen contamination of produce was the leading cause of foodborne illnesses and outbreaks associated with a single-ingredient commodity between 2004 and 2013 (Fischer et al, 2015)
The importance of foodborne illness caused by contamination of produce by Salmonella spp. and the prevalence of contamination associated with leafy greens (DeWaal et al, 2008) led us to investigate the molecular mechanisms allowing Salmonella spp. to use this alternate host for survival
As apoplastic populations of human pathogenic bacteria in lettuce are a potential risk for foodborne illnesses due to persistence from production to consumption, we directed our focus on the bacterial internalization into leaves through stomata and endophytic survival

Summary

Introduction

Human pathogen contamination of produce was the leading cause of foodborne illnesses and outbreaks associated with a single-ingredient commodity between 2004 and 2013 (Fischer et al, 2015). Once on the leaf surface, bacteria are faced with harsh conditions, such as UV irradiation, low nutrient and water availability, and unfavorable weather (Hirano and Upper, 1983; Lindow and Brandl, 2003) Bacteria may escape these conditions by attaching to the leaf surface and forming biofilms (Kroupitski et al, 2009) or by transitioning to an endophytic lifestyle through internalization into the leaf extracellular space (i.e., apoplast) via natural pores or wounds (Kroupitski et al, 2009; Critzer and Doyle, 2010; Roy et al, 2013). This suggests that internalization trades one challenge for another (i.e., those of the phylloplane for those of the apoplast), and only bacteria that can cope with these challenges will be able to colonize leaves successfully

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Conclusion