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Distribution of Salmonella Typhimurium in romaine lettuce leaves
Abstract
Leafy greens are occasionally involved in outbreaks of enteric pathogens. In order to control the plant contamination it is necessary to understand the factors that influence enteric pathogen-plant interactions. Attachment of Salmonella enterica serovar typhimurium to lettuce leaves has been demonstrated before; however, only limited information is available regarding the localization and distribution of immigrant Salmonella on the leaf surface. To extend our knowledge regarding initial pathogen-leaf interactions, the distribution of green-fluorescent protein-labeled Salmonella typhimurium on artificially contaminated romaine lettuce leaves was analyzed. We demonstrate that attachment of Salmonella to different leaf regions is highly variable; yet a higher attachment level was observed on leaf regions localized close to the petiole (7.7 log CFU g(-1)) compared to surfaces at the far-end region of the leaf blade (6.2 log CFU g(-1)). Attachment to surfaces located at a central leaf region demonstrated intermediate attachment level (7.0 log CFU g(-1)). Salmonella displayed higher affinity toward the abaxial side compared to the adaxial side of the same leaf region. Rarely, Salmonella cells were also visualized underneath stomata within the parenchymal tissue, supporting the notion that this pathogen can also internalize romaine lettuce leaves. Comparison of attachment to leaves of different ages showed that Salmonella displayed higher affinity to older compared to younger leaves (1.5 log). Scanning electron microscopy revealed a more complex topography on the surface of older leaves, as well as on the abaxial side of the examined leaf tissue supporting the notion that a higher attachment level might be correlated with a more composite leaf landscape. Our findings indicate that initial attachment of Salmonella to romaine lettuce leaf depends on multiple plant factors pertaining to the specific localization on the leaf tissue and to the developmental stage of the leaf.
... The higher bacterial recovery from the abaxial than the adaxial side of leaves corroborates previous studies, where the recovery of E. coli O157:H7 at 2-3 days after inoculation was significantly higher when lettuce was inoculated on the abaxial side of leaves as opposed to the adaxial side (29,34,35). The leaf surface properties that may promote better bacterial adhesion or persistence on the abaxial leaf side possibly include increased stomatal size and density (19,36) and higher physicochemical heterogeneity (35). ...
... The higher bacterial recovery from the abaxial than the adaxial side of leaves corroborates previous studies, where the recovery of E. coli O157:H7 at 2-3 days after inoculation was significantly higher when lettuce was inoculated on the abaxial side of leaves as opposed to the adaxial side (29,34,35). The leaf surface properties that may promote better bacterial adhesion or persistence on the abaxial leaf side possibly include increased stomatal size and density (19,36) and higher physicochemical heterogeneity (35). In our study, leaf side affected recovery only for strain 2705C and only when cultivar was differentiated, suggesting a weak effect of leaf side in the pathogen-cultivar systems used. ...
The varied choice of bacterial strain, plant cultivar, and method used to inoculate, retrieve, and enumerate Escherichia coli O157:H7 from live plants could affect comparability among studies evaluating lettuce–enterobacterial interactions. Cultivar, bacterial strain, incubation time, leaf side inoculated, and sample processing method were assessed for their influence in recovering and quantifying E. coli O157:H7 from live Romaine lettuce. Cultivar exerted the strongest effect on E. coli O157:H7 counts, which held up even when cultivar was considered in interactions with other factors. Recovery from the popularly grown green Romaine “Rio Bravo” was higher than from the red variety “Outredgeous.” Other modulating variables were incubation time, strain, and leaf side inoculated. Sample processing method was not significant. Incubation for 24 hours post-lettuce inoculation yielded greater counts than 48 hours, but was affected by lettuce cultivar, bacterial strain, and leaf side inoculated. Higher counts obtained for strain EDL933 compared to a lettuce outbreak strain 2705C emphasized the importance of selecting relevant strains for the system being studied. Inoculating the abaxial side of leaves gave higher counts than adaxial surface inoculation, although this factor interacted with strain and incubation period. Our findings highlight the importance of studying interactions between appropriate bacterial strains and plant cultivars for more relevant research results, and of standardizing inoculation and incubation procedures. The strong effect of cultivar exerted on the E. coli O157:H7-lettuce association supports the need to start reporting cultivar information for illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.
IMPORTANCE
The contamination of Romaine lettuce with Escherichia coli O157:H7 has been linked to multiple foodborne disease outbreaks, but variability in the methods used to evaluate E. coli O157:H7 association with live lettuce plants complicates the comparability of different studies. In this study, various experimental variables and sample processing methods for recovering and quantifying E. coli O157:H7 from live Romaine lettuce were assessed. Cultivar was found to exert the strongest influence on E. coli O157:H7 retrieval from lettuce. Other modulating factors were bacterial incubation time on plants, strain, and leaf side inoculated, while sample processing method had no impact. Our findings highlight the importance of selecting relevant cultivars and strains, and of standardizing inoculation and incubation procedures, in these types of assessments. Moreover, results support the need to start reporting cultivars implicated in foodborne illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.
... Salmonella is a member of the Enterobacteriaceae family, which possesses a conserved genomic backbone (Alnajar and Gupta, 2017). Enterobacteriaceae includes human and plant pathogens, both of which can attach to and internalize into plant tissues (Brandl and Mandrell, 2002;Kroupitski et al., 2011;Roy et al., 2013). However, enterobacterial human pathogens like S. enterica are not known to contain plant-related virulence factors, such as phytotoxins and cell wall-degrading enzymes, capable of causing symptoms or disease on plants (Toth et al., 2006). ...
... In this study, we evaluated the capacity of 14 S. enterica isolates from 10 different serovars, collected from plant and animal sources, to persist and compete for survival in the leaf apoplast of two lettuce cultivars that have contrasting responses to bacterial colonization. The lettuce leaf apoplast was utilized as S. enterica can internalize into and reside in this habitat (Kroupitski et al., 2011;Roy and Melotto, 2019). Bacterial fitness competition assays were performed using clones distinguished by barcodes in their genomes (Santiviago et al., 2009;Silva-Valenzuela et al., 2016;Porwollik et al., 2018). ...
Multiple Salmonella enterica serovars and strains have been reported to be able to persist inside the foliar tissue of lettuce (Lactuca sativa L.), potentially resisting washing steps and reaching the consumer. Intraspecies variation of the bacterial pathogen and of the plant host can both significantly affect the outcome of foliar colonization. However, current understanding of the mechanisms underlying this phenomenon is still very limited. In this study, we evaluated the foliar fitness of 14 genetically barcoded S. enterica isolates from 10 different serovars, collected from plant and animal sources. The S. enterica isolates were vacuum-infiltrated individually or in pools into the leaves of three- to four-week-old lettuce plants. To estimate the survival capacity of individual isolates, we enumerated the bacterial populations at 0- and 10- days post-inoculation (DPI) and calculated their net growth. The competition of isolates in the lettuce apoplast was assessed through the determination of the relative abundance change of barcode counts of each isolate within pools during the 10 DPI experimental period. Isolates exhibiting varying apoplast fitness phenotypes were used to evaluate their capacity to grow in metabolites extracted from the lettuce apoplast and to elicit the reactive oxygen species burst immune response. Our study revealed that strains of S. enterica can substantially differ in their ability to survive and compete in a co-inhabited lettuce leaf apoplast. The differential foliar fitness observed among these S. enterica isolates might be explained, in part, by their ability to utilize nutrients available in the apoplast and to evade plant immune responses in this niche.
... Irreversible adhesion of S. Typhimurium on the plants' edible parts was demonstrated on lettuce, basil, parsley, peppers, tomatoes, and more (Lapidot and Yaron, 2009;Kroupitski et al., 2011;Shaw et al., 2011, Kisluk andYaron, 2012). The attachment to the phyllosphere may be followed by either colonization and biofilm formation on the surface (Lapidot et al., 2006), entry into the host tissue (Kroupitski et al., 2011), or invasion into the plant cells (Schikora et al., 2008;Shirron and Yaron, 2011). ...
... Irreversible adhesion of S. Typhimurium on the plants' edible parts was demonstrated on lettuce, basil, parsley, peppers, tomatoes, and more (Lapidot and Yaron, 2009;Kroupitski et al., 2011;Shaw et al., 2011, Kisluk andYaron, 2012). The attachment to the phyllosphere may be followed by either colonization and biofilm formation on the surface (Lapidot et al., 2006), entry into the host tissue (Kroupitski et al., 2011), or invasion into the plant cells (Schikora et al., 2008;Shirron and Yaron, 2011). Endophytic survival helps the bacteria to escape from the hostile and instable environment on the leaf surface, but forces the bacteria to cope with new challenges like the plant defense response. ...
Salmonella enterica is one of the most common human pathogens associated with fresh produce outbreaks. The present study suggests that expression of BcsZ, one of the proteins in the bcs complex, enhances the survival of Salmonella Typhimurium on parsley. BcsZ demonstrated glucanase activity with the substrates carboxymethylcellulose and crystalline cellulose, and was responsible for a major part of the S. Typhimurium CMCase activity. Moreover, there was constitutive expression of BcsZ, which was also manifested after exposure to plant polysaccharides and parsley-leaf extract. In an in-planta model, overexpression of BcsZ significantly improved the epiphytic and endophytic survival of S. Typhimurium on/in parsley leaves compared with the wild-type strain and bcsZ null mutant. Interestingly, necrotic lesions appeared on the parsley leaf after infiltration of Salmonella overexpressing BcsZ, while infiltration of the wild-type S. Typhimurium did not cause any visible symptoms. Infiltration of purified BcsZ enzyme, or its degradation products also caused symptoms on parsley leaves. We suggest that the BcsZ degradation products trigger the plant’s defense response, causing local necrotic symptoms. These results indicate that BcsZ plays an important role in the Salmonella-plant interactions, and imply that injured bacteria may take part in these interactions.
... Lettuce genotypes have extensive variations in leaf traits including the content of surface phenolics, proteins, wax, and sugars; contact angle; and stomatal density (Hunter et al., 2015). These properties have been shown to facilitate or hamper the leaf attachment of human pathogenic bacteria (Golberg et al., 2011;Kroupitski et al., 2011;Hunter et al., 2015). In this study, we FIGURE 7 | Escherichia coli O157:H7 (A) and Salmonella enterica Typhimurium 14028s (B) persistence varies in 3.5-to 4-week-old plants of lettuce genotypes after syringe inoculation. ...
... The lack of correlation between these variables might be due to the low number of genotypes used in our study. Nevertheless, Kroupitski et al. (2011) also observed no relationship between differential attachment of S. Typhimurium SL1344 on abaxial and adaxial leaf surfaces and the level of stomatal density in romaine lettuce. Overall, the FIGURE 8 | Reactive oxygen species (ROS) burst induced by Escherichia coli O157:H7 and Salmonella enterica Typhimurium 14028s varies among the lettuce genotypes. ...
Fresh produce contaminated with human pathogens may result in foodborne disease outbreaks that cause a significant number of illnesses, hospitalizations, and death episodes affecting both public health and the agribusiness every year. The ability of these pathogens to survive throughout the food production chain is remarkable. Using a genetic approach, we observed that leaf colonization by Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium 14028s) and Escherichia coli O157:H7 was significantly affected by genetic diversity of lettuce (Lactuca sativa L. and L. serriola L.). In particular, there was a significant variation among 11 lettuce genotypes in bacterial attachment, internalization, and apoplastic persistence after surface- and syringe-inoculation methods. We observed a significant correlation of the bacterial leaf internalization rate with stomatal pore traits (width and area). Moreover, bacterial apoplastic populations significantly decreased in 9 out of 11 lettuce genotypes after 10 days of surface inoculation. However, after syringe infiltration, populations of E. coli O157:H7 and S. Typhimurium 14028s showed positive, neutral, or negative net growth in a 10-day experimental period among seedlings of different lettuce types. The relative ability of the bacteria to persist in the apoplast of lettuce genotypes after syringe inoculation was minimally altered when assessed during a longer period (20 days) using 3.5- to 4-week-old plants. Interestingly, contrasting bacterial persistence in the lettuce genotypes Red Tide and Lollo Rossa was positively correlated with significant differences in the level of reactive oxygen species burst and callose deposition against S. Typhimurium 14028s and E. coli O157:H7 which are related to plant defense responses. Overall, we characterized the genetic diversity in the interaction between lettuce genotypes and enterobacteria S. Typhimurium 14028s and E. coli O157:H7 and discovered that this genetic diversity is linked to variations in plant immune responses towards these bacteria. These results provide opportunities to capitalize on plant genetics to reduce pathogen contamination of leaves.
... The ability of Salmonella to adhere to lettuce leaves was reported by several studies (Wei et al., 2006;Patel and Sharma, 2010;Kroupitski et al., 2011;Lima et al., 2013). Results similar to the ones obtained in this study were found by Kroupitski et al. (2011), who found 7.0 log CFU of S. Typhimurium on the central region of lettuce leaves. ...
... The ability of Salmonella to adhere to lettuce leaves was reported by several studies (Wei et al., 2006;Patel and Sharma, 2010;Kroupitski et al., 2011;Lima et al., 2013). Results similar to the ones obtained in this study were found by Kroupitski et al. (2011), who found 7.0 log CFU of S. Typhimurium on the central region of lettuce leaves. Also, Lima et al. (2013) demonstrated that the count of S. Enteritidis cells that adhered to lettuce leaves differed (P < 0.05) between the hydroponic and conventional systems, reaching 5.2 ± 0.56 and 4.6 ± 0.26, respectively. ...
Salmonella Enteritidis SE86 is an important foodborne pathogen in Southern Brazil and it is able to produce a biosurfactant. However, the importance of this compound for the microorganism is still unknown. This study aimed to investigate the influence of biosurfactant produced by S. Enteritidis SE86 on the adherence to slices of lettuce leaves and on the resistance to sanitizers. First, S. Enteritidis SE86 was inoculated on lettuce leaves in order to determine the amount of biosurfactant produced. Subsequently, S. Enteritidis SE86 was inoculated on lettuce leaves, with and without the biosurfactant, and the adherence and bacterial resistance to different sanitization methods were evaluated. S. Enteritidis SE86 produced biosurfactant after 16 hours (emulsification index of 11 to 52.15%) and showed greater adherence capability and resistance to sanitization methods when the compound was present. The scanning electron microscopy demonstrated that S. Enteritidis was able to adhere, form lumps, and invade the lettuce leaves stomata in the presence of biosurfactant. Results indicated that the biosurfactant produced by S. Enteritidis SE86 contributed to the adherence and increased the resistance to sanitizers when the microorganism was present on lettuce leaves.
... Leaf attachment assay A leaf attachment assay was performed following the method described by Kroupitski et al. [44] with minor The experiment was conducted in triplicates. ...
processes. The effects of light, oxygen, or voltage (LOV) and blue light (BL) photosensory proteins on the virulence and pathogenic features of plant bacterial pathogens require further exploration. In this study, we identified LOV protein (Pc-LOV1) from Pseudomonas cichorii JBC1 (PcJBC1) and characterized its function using LOV1-deficient mutant (JBC1 Δlov1 ). The recombinant Pc-LOV1 protein showed absorption peaks at 448, 425, and 475 nm in the dark, which is a typical feature of oxidized flavin chromophores. BL negatively influenced the virulence of PcJBC1 and the virulence of JBC1 Δlov1 increased irrespective of BL, indicating that Pc-LOV1 negatively regulates PcJBC1 virulence. Pc-LOV1 and BL positively regulated the biological features relevant to infection processes, such as adhesion to the plant surface and biofilm formation. In contrast, swarming motility, exopolysaccharide and siderophore production were negatively controlled. Gene expression and in silico analysis of the interacting proteins supported the modulation of bacterial features. Overall, our results suggest that the LOV photosensory system plays crucial roles in the adaptive responses and pathogenicity of the bacterial pathogen PcJBC1. The roles of other photoreceptors, sensing of other light wavelengths, and signal networking require further investigation.
... The ability of the JBC1 DdnaJ , JBC1 JDpvdS , and their complemented strains to colonize the epiphytic surface of the leaves was investigated following the procedures by Kroupitski et al. [30], with minor modifications. Briefly, overnight cultures of each strain were resuspended in sDW at a concentration of 1 Â 10 8 CFU/mL in 50 mL falcon tubes. ...
Bacterial plant pathogens must cope with various environmental conditions and defenses from their hosts for colonization and infection. Heat shock proteins (HSPs) play critical roles in a variety of cellular processes, such as the maintenance of cellular homeostasis in response to environmental stress. However, the significance of HSP40 family protein DnaJ in virulence of plant pathogenic bacteria has not yet been explored. To elucidate the function of DnaJ in Pseudomonas cichorii JBC1 (PcJBC1) virulence, we generated dnaJ-deficient (JBC1ΔdnaJ) mutant using CRISPR-CAS9. The disease severity by JBC1ΔdnaJ was significantly reduced compared with wild-type (WT) and dnaJ-complemented (JBC1Δdnaj+pdnaJ) strain. The defect of DnaJ suppressed siderophore production, extracellular DNA (eDNA) release, biofilm formation, and swarming motility and made the strain sensitive to stresses such as heat and H2O2. The supplementation of eDNA recovered the amount of biofilm formation by JBC1ΔdnaJ. Our results indicate that DnaJ is a key plyer in the survival and colonization of bacterial plant pathogens on plant surfaces as well as bacterial responses to abiotic and biotic stresses, which are determinative to cause disease. These findings can broaden our understanding of plant and bacterial pathogen interactions.
... Romaine and iceberg lettuce are cored manually by workers in the field, and together with the close proximity to coring tools and soil, the risk of cross contamination is higher (Erickson, 2012). Minimal processing techniques such as cleaning the cut edges with current sanitizing washes and agents are not more effective than using tap water and do not completely reduce the microbial load on lettuce to prevent cross-contamination (Kroupitski et al., 2011;Van der Linden et al., 2016). Currently, the most popular method for washing lettuce and leafy vegetables is using chlorine at 50 to 200 ppm for up to 5 min (Meireles et al., 2016;Van Haute et al., 2013). ...
The antimicrobial potential of switchgrass extractives (SE) was evaluated on cut lettuce leaves and romaine lettuce in planta, using rifampicin‐resistant Escherichia coli O157:H7 and Salmonella Typhimurium strain LT2 as model pathogens. Cut lettuce leaves were swabbed with E. coli O157:H7 or S. Typhimurium followed by surface treatment with 0.8% SE, 0.6% sodium hypochlorite, or water for 1 to 45 min. For in planta studies, SE was swabbed on demarcated leaf surfaces either prior to or after inoculation of greenhouse‐grown lettuce with E. coli O157:H7 or S. Typhimurium; the leaf samples were collected after 0, 24, and 48 h of treatment. Bacteria from inoculated leaves were enumerated on tryptic soy agar plates (and also on MacConkey's and XLT4 agar plates), and the recovered counts were statistically analyzed. Cut lettuce leaves showed E. coli O157:H7 reduction between 3.25 and 6.17 log CFU/leaf, whereas S. Typhimurium reductions were between 2.94 log CFU/leaf and 5.47 log CFU/leaf depending on the SE treatment durations, from initial levels of ∼7 log CFU/leaf. SE treatment of lettuce in planta, before bacterial inoculation, reduced E. coli O157:H7 and S. Typhimurium populations by 1.88 and 2.49 log CFU after 24 h and 3 h, respectively. However, SE treatment after bacterial inoculation of lettuce plants decreased E. coli O157:H7 populations by 3.04 log CFU (after 0 h) with negligible reduction of S. Typhimurium populations. Our findings demonstrate the potential of SE as a plant‐based method for decontaminating E. coli O157:H7 on lettuce during pre‐ and postharvest stages in hurdle approaches.
... Surfactants have been used to facilitate the removal of microorganisms from fresh produce surfaces, primarily due to their ability to reduce the surface tension of the wash solution and enhance the spread across a surface [68]. Specifically, Huang et al. (2018) evaluated the influence of surfactants on a detachment of Escherichia coli O157:H7 on lettuce leaves, a food which, it has been hypothesized, has been commonly implicated in foodborne outbreaks and recall events in part due to its complex surface topography to enable microbial attachment [69]. The study found that the use of 0.05% and 0.1% Tween 20 in the wash water solution facilitated bacterial removal by up to 2.13-log CFU/cm 2 across the leaf surface, with log reduction increasing with the surfactant concentration [70]. ...
Apples are the most consumed fruit in the United States and have recently been shown to exhibit some vulnerability to contamination across the supply chain. It is unclear what role a fruit microbiome analysis may serve in future food safety programs interested in understanding changes in the product and the processing environment. Ultimately, sample integrity is key if any of these approaches are to be employed; low microbial loads on apple surfaces, the inability to sample the entire surface, and inefficiency of removal may act as barriers to achieving high-quality DNA. As such, the objective of this study was to identify a reproducible method to concentrate and quantify bacterial and fungal DNA from fresh apple surfaces. Five methods were evaluated: two variations of wash solutions for bath sonication, wash filtration, epidermis excision, and surface swabbing. Epidermis excision returned the highest mean DNA quantities, followed by the sonicated washes and wash filtration. Surface swabbing was consistently below the limit of detection. Based on the quantity of host DNA contamination in surface excision, the sonicated wash solution containing a surfactant presents the greatest opportunity for consistent, high-yielding DNA recovery from the entire apple surface.
... Previously, plants were thought to be passive vectors for human pathogens, but recent studies showed that S. enterica can induce plant defense responses (Meng et al., 2013;Garcia and Hirt, 2014;Melotto et al., 2014;Oblessuc et al., 2020). Intriguingly, although the mechanism is not fully understood, this bacterium can overcome plant defense (Roy et al., 2013;Wahlig et al., 2019) and survive for weeks inside diverse plants species, including lettuce (Lactuca sativa L.) (Islam et al., 2004;Kroupitski et al., 2009Kroupitski et al., , 2011Jechalke et al., 2019;. These findings have prompted further research on the interaction between plants and human pathogens. ...
Salmonella enterica is an enterobacterium associated with numerous foodborne illnesses worldwide. Leafy greens have been a common vehicle for disease outbreaks caused by S. enterica. This human pathogen can be introduced into crop fields and potentially contaminate fresh produce. Several studies have shown that S. enterica can survive for long periods in the plant tissues. Often, S. enterica population does not reach high titers in leaves; however, it is still relevant for food safety due to the low infective dose of the pathogen. Thus, laboratory procedures to study the survival of S. enterica in fresh vegetables should be adjusted accordingly. Here, we describe a protocol to assess the population dynamics of S. enterica serovar Typhimurium 14028s in the leaf apoplast of three cultivars of lettuce (Lactuca sativa L.). By comparing a range of inoculum concentrations, we showed that vacuum infiltration of a bacterium inoculum level in the range of 3.4 Log CFU ml–1 (with a recovery of approximately 170 cells per gram of fresh leaves 2 h post inoculation) allows for a robust assessment of bacterial persistence in three lettuce cultivars using serial dilution plating and qPCR methods. We anticipate that this method can be applied to other leaf–human pathogen combinations in an attempt to standardize the procedure for future efforts to screen for plant phenotypic variability, which is useful for breeding programs.
... Nevertheless, given the recurrence of food-borne illness outbreaks linked to produce (Figure 1), the ability of enteric pathogens to multiply and survive as epiphytes and endophytes implies that particular plant phenotypes and genotypes can affect their fitness in the plant habitat (Table 1). For example, the composition of substrates available on fruit and leaf surfaces as well as in their internal tissue (Brandl and Amundson, 2008;Marvasi et al., 2014b;Crozier et al., 2016;Han and Micallef, 2016); the density of trichomes, stomata, and veins (Barak et al., 2011;Kroupitski et al., 2011;Macarisin et al., 2013;Jacob and Melotto, 2020), which harbor larger pools of substrates than other areas of leaves; and the physical and chemical composition of the cuticle layer on various parts of the plant (Lima et al., 2013;Hunter et al., 2015), which affects water dispersal and hence, water and nutrient availability to microbial inhabitants (Marcell and Beattie, 2002), may all be relevant traits to investigate in plant breeding efforts for their effect on enteric pathogen colonization. ...
An increasing global population demands a continuous supply of nutritious and safe food. Edible products can be contaminated with biological (e.g., bacteria, virus, protozoa), chemical (e.g., heavy metals, mycotoxins), and physical hazards during production, storage, transport, processing, and/or meal preparation. The substantial impact of foodborne disease outbreaks on public health and the economy has led to multidisciplinary research aimed to understand the biology underlying the different contamination processes and how to mitigate food hazards. Here we review the knowledge, opportunities, and challenges of plant breeding as a tool to enhance the food safety of plant-based food products. First, we discuss the significant effect of plant genotypic and phenotypic variation in the contamination of plants by heavy metals, mycotoxin-producing fungi, and human pathogenic bacteria. In addition, we discuss the various factors (i.e., temperature, relative humidity, soil, microbiota, cultural practices, and plant developmental stage) that can influence the interaction between plant genetic diversity and contaminant. This exposes the necessity of a multidisciplinary approach to understand plant genotype × environment × microbe × management interactions. Moreover, we show that the numerous possibilities of crop/hazard combinations make the definition and identification of high-risk pairs, such as Salmonella-tomato and Escherichia coli-lettuce, imperative for breeding programs geared toward improving microbial safety of produce. Finally, we discuss research on developing effective assays and approaches for selecting desirable breeding germplasm. Overall, it is recognized that although breeding programs for some human pathogen/toxin systems are ongoing (e.g., Fusarium in wheat), it would be premature to start breeding when targets and testing systems are not well defined. Nevertheless, current research is paving the way toward this goal and this review highlights advances in the field and critical points for the success of this initiative that were discussed during the Breeding Crops for Enhanced Food Safety workshop held 5–6 June 2019 at University of California, Davis.
... Recovery of E. coli from the irrigated "Annapolis" lettuce with the highest stomata density was not significantly different from the E. coli recovered from the "Celinet" and "Coastline" lettuce, except for the results on 2 dpi of the second week. Likewise, Kroupitski, Pinto, Belausov, and Sela (2011) observed higher bacterial attachment on the abaxial side of the leaf, and since there was no difference in the number of stomata on both sides of the same leaf, results suggested that other morphological factors could influence the attachment of S. Typhimurium to the "Romaine" lettuce. Besides stomatal density, other leaf structural and chemical traits such as thickness of the leaf, glandular trichome density, as well as total phenolic, phosphorus, and water contents were reported to affect the bacterial attachment and colonization on vegetable leaf (Brandl, 2006;Heaton & Jones, 2008;Leveau, 2009). ...
Effect of irrigation with groundwater (GW), primary‐treated wastewater (PTWW), secondary‐treated wastewater (STWW), and roof‐collected rainwater (RCR) on the microbial quality of lettuce cultivars “Annapolis,” “Celinet,” and “Coastline” grown in high tunnel was investigated. Lettuce plants were spray irrigated with irrigation waters once a week for 2 weeks and analyzed for indicator and pathogenic bacteria. PTWW irrigation resulted in the highest Escherichia coli recovery on the lettuce plants (4.7 log MPN/g) as compared to irrigation with other three waters on 0 day postirrigation (dpi). Lettuce cultivars affected the bacterial die‐off rate, where E. coli populations reduced the most by 1.5 log MPN/g on “Annapolis” lettuce on 2 dpi. The STWW and RCR irrigation did not significantly influence indicator bacterial populations on lettuce as compared to GW irrigation. The STWW and RCR containing low populations of indicator bacteria may be suitable for lettuce irrigation in Mid‐Atlantic area without affecting its microbial quality.
... [14] However bacterial internalization in vegetable leaves has been reported for lettuce, which makes disinfection inefficient. [11,15] ...
Background: Salad which is served at restaurants in uncooked form can be source of bacterial contamination.Aim: To assess the microbiological quality of lettuce (Lactuca sativa) salads sold in two restaurants in Kigali, Rwanda.Methods: Lettuce samples were collected using aseptic techniques and immediately analysed using standard media for E. coli, Salmonella, Shigella and Staphyloccocus aureus besides total aerobic count.Results: The number of total aerobic bacteria ranged from 1.15 x104 to 2.87x104 cfu/ml for restaurant 1 and from 1.91x104 to 2.13x105cfu/ml for restaurant 2. Staphyloccoci count ranged from 3.6x102 to 1.173x103 cfu/ml for restaurant 1 and from 1.9 x102 to 3.2 x102 cfu/ml for restaurant 2. Gram negative bacteria count ranged from 8.3 x102 to 1.03 x105 cfu/ml for restaurant 1 and from 5.05 x102 to 2.30 x105 cfu/ml for restaurant 2. Salmonella and Shigella count ranged from 2.54x103 to 2.04x104 cfu/ml for restaurant 1 and from 3.65x103 to 2.5x104 cfu/ml for restaurant 2. E. coli was found in 75% of the sample from restaurant 1 and 100% of the sample from restaurant 2.Conclusion: Improvement in hygiene is needed during preparation and handling of lettuce salad in these restaurants.Keywords: Lactuca sativa, microbiological quality, restaurants, Rwanda
... Four sterile lettuce samples were transferred to Falcon tubes and submerged into 10 mL of an E. coli suspension (approximately 10 8 CFU•mL −1 ) for 60 min at room temperature [45]. After this period, the cell suspension was discarded and the lettuce samples were immediately washed twice for 1 min with sterile deionized water to remove the bacteria that had not adhered to the lettuce [45,46]. The lettuce samples were then immersed in 30 mL of the intended concentration of sodium hypochlorite or peracetic acid for 5 min. ...
Foodborne outbreaks due to the consumption of ready-to-eat vegetables have increased worldwide, with Escherichia coli (E. coli) being one of the main sources responsible. Viable but nonculturable bacteria (VBNC) retain virulence even after some disinfection procedures and constitute a huge problem to public health due to their non-detectability through conventional microbiological techniques. Flow cytometry (FCM) is a promising tool in food microbiology as it enables the distinction of the different physiological states of bacteria after disinfection procedures within a short time. In this study, samples of lettuce inoculated with E. coli were subject to disinfection with sodium hypochlorite at free chlorine concentrations of 5, 10, 25, 50, and 100 mg·L−1 or with 35% peracetic acid at concentrations of 5, 10, 25, and 50 mg·L−1. The efficiency of these disinfectants on the viability of E. coli in lettuce was evaluated by flow cytometry with LIVE/DEAD stains. Results from this study suggest that FCM can effectively monitor cell viability. However, peracetic acid is more effective than sodium hypochlorite as, at half the concentration, it is enough to kill 100% of bacteria and always induces a lower percentage of VBNC. Finally, we can conclude that the recommended levels of chemical disinfectants for fresh fruit and vegetables are adequate when applied in lettuce. More importantly, it is possible to ensure that all cells of E. coli are dead and that there are no VBNC cells even with lower concentrations of those chemicals. These results can serve as guidance for lettuce disinfection, improving quality and the safety of consumption.
... Aerosol spray may also facilitate deposition of contaminated wastewater on the edible parts of the crop. Surface contamination of these has been reported as a major means of transmission of diseases through consumption (90)(91)(92)(93). Additionally, internalization of pathogens in different vegetables has been documented (94)(95)(96)(97)(98), which could be another route Table 1, where further information is provided. ...
The use of partially treated and untreated wastewater for irrigation is beneficial in agriculture but may be associated with human health risks. Reports from different locations globally have linked microbial outbreaks with agricultural reuse of wastewater. This article reviews the epidemiological evidence and health risks associated with this practice, aiming toward evidence-based conclusions. Exposure pathways that were addressed in this review included those relevant to agricultural workers and their families, consumers of crops, and residents close to areas irrigated with wastewater (partially treated or untreated). A meta-analysis gave an overall odds ratio of 1.65 (95% CI: 1.31, 2.06) for diarrheal disease and 5.49 (95% CI: 2.49, 12.10) for helminth infections for exposed agricultural workers and family members. The risks were higher among children and immunocompromised individuals than in immunocompetent adults. Predominantly skin and intestinal infections were prevalent among individuals infected mainly via occupational exposure and ingestion. Food-borne outbreaks as a result of crops (fruits and vegetables) irrigated with partially or untreated wastewater have been widely reported. Contamination of crops with enteric viruses, fecal coliforms, and bacterial pathogens, parasites including soil-transmitted helminthes (STHs), as well as occurrence of antibiotic residues and antibiotic resistance genes (ARGs) have also been evidenced. The antibiotic residues and ARGs may get internalized in crops along with pathogens and may select for antibiotic resistance, exert ecotoxicity, and lead to bioaccumulation in aquatic organisms with high risk quotient (RQ). Appropriate mitigation lies in adhering to existing guidelines such as the World Health Organization wastewater reuse guidelines and to Sanitation Safety Plans (SSPs). Additionally, improvement in hygiene practices will also provide measures against adverse health impacts.
... In this work, 3 min of plasma treatment resulted in a reduction of at least 2.6-log of S. Typhimurium from a contaminated lettuce surface (Fig. 3). Variations in decontamination potential depend on a number of factors such as surface roughness, width, moistness of the treated sample, type of target microorganism and type of plasma-discharged source (Kroupitski et al., 2011;Niemira, 2014). To obtain a fair measurement the bacterial suspension was inoculated in the back side of the lettuce surface. ...
The potential of micro-plasma discharged water (m-PDW) treatment on reduction of Salmonella Typhimurium ATCC 14028 from the surface of lettuce leaves was evaluated. The surface of iceberg lettuce and red leaf lettuce pieces (3 cm × 3 cm) was inoculated with a S. Typhimurium suspension. The measured S. Typhimurium reductions were 3.0-log CFU/piece and 2.6-log CFU/piece for iceberg lettuce and red leaf lettuce, respectively for a 3-min treatment. The overall reductions measured for the levels of kaempferol and quercetin in both types of lettuce were within the experimental error for a 3-min plasma treatment. There was no significant change in color (p > 0.05) for plasma operations of the same duration. Leaching of water contributed to a greater extent towards physiochemical changes such as color constraints, kaempferol and quercetin content occurred for the treated lettuce samples, although the overall physicochemical quality changes measured in this study remains in the satisfactory level.
... Variability in bacterial phyllosphere populations has been correlated with leaf position, age and nutrient availability as well as the accessibility or exposure of leaves to the deposition of airborne microbiota, microclimates, growing season, leaf orientation, plant structure and the physicochemical condition of leaves (Mew and Kennedy, 1982;Plummer et al., 1992;Jacques et al., 1995;Ong et al., 1999;Redford and Fierer 2009;Lindow and Brandl, 2003;Aruscavage et al., 2006;Monier and Lindow, 2005;Redford et al., 2010;Jackson and Denney, 2011;Kroupitski et al., 2011;Burch et al., 2016). Rastogi et al. (2012) evaluated the extent and presumptive sources of variability on the phyllosphere bacterial community composition on Romaine lettuce as a function of time, spatial separation within and between commercial fields, and seasonal environmental and geospatial differences. ...
... Also, there is differences in attachment Salmonella typhimurium to plant leaves and it depends on plant growing stage, better attachment is at older than at youger leaves [45]. ...
Contamination of vegetables and fruits is the result of presence of human pathogen bacteria which can contaminate products in any part of production chain. There is an evidence of presence of: Salmonella spp. on the fresh vegetables and Salmonellosis is connected with tomato, sprouts, cantaloupe etc.
The goal of this research is transmission of pathogen bacteria from irrigation water to plants and studying/monitoring the ability of the Salmonella spp. to colonize the surface and interior (endophytic colonization) of root at different vegetable species.
Transmission of three Salmonella spp. strains from irrigation water to plants, as well as colonization of plants by these bacteria was investigated by using Fluorescence In Situ Hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM).
All tested Salmonella spp. strains showed ability to more or less colonize the surface and interior niches of the root, stem and leaf of the investigated plant species. These bacteria also were found in plant cells cytoplasm, although the mechanism of their entrance has not been clarified yet.
... As specific decontamination of S. enterica from fruits and vegetables is practically impossible, contamination should be avoided or reduced. Unfortunately, contaminations of produce with S. enterica can occur along the whole production chain and one possible source is the soil (Guo et al., 2002;Klerks et al., 2007;Barak et al., 2011;Kroupitski et al., 2011;Olaimat and Holley, 2012). Occurrence of S. enterica in soil can originate from irrigation or flooding water, organic fertilizers, feces or plant residues plowed into soil after harvest (Semenov et al., 2010;Jacobsen and Bech, 2012;Olaimat and Holley, 2012). ...
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.
... While S. Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Senftenberg were efficient, other serovars including Salmonella enterica serovar Agona, Salmonella enterica serovar Heidelberg or Salmonella enterica serovar Arizonae showed less attachment [67]. Clear differences in attachment were also observed in leaves of different age, for example, S. Typhimurium showed a better attachment to older compared to younger lettuce leaves [68]. Additionally, S. enterica serovars were reported to actively move toward plant roots, attracted by root exudates [69]. ...
... Finding SD-GII.4P in stomata in this study is interesting as stomata might provide protection to viruses from liquid disinfectants or simple washing. It is common to find bacteria in stomata because of the move ability of bacteria (Xicohtencatl-Cortes et al., 2009;Golberg et al., 2011;Kroupitski et al., 2011). Wei et al reported that MNV could be found in stomata, at the cut edges and on leaf surfaces of romaine lettuce (Wei et al., 2010). ...
Human Noroviruses (HuNoVs) are the main cause of non-bacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein on bacterial surface and used it as a new strategy to explore interaction between HuNoV protein and receptor candidates from romaine lettuce. The surface-displayed HuNoV proteins were confirmed on the surface of the transformed bacteria by an immunofluorescence assay. The distribution patterns of the surface-displayed HuNoV proteins in romaine lettuce were identified through a confocal immunofluorescence assay. The surface-displayed HuNoV proteins could be found in the stomata, and the surfaces of vein and leaf of romaine lettuce. The surface-displayed HuNoV proteins could be captured by an ELISA assay utilizing extract from leaf (LE) or vein (VE). The binding of the surface-displayed HuNoV proteins to LE or VE could be competitively blocked by histo-blood group antigens from human saliva. In addition, the binding of the surface-displayed HuNoV proteins to LE or VE could also be attenuated by heat denaturation of lettuce proteins, and abolished by oxidation of lettuce carbohydrates. The results indicated that histo-blood group antigen-like molecules in LE or VE were involved in the binding of the surface-displayed HuNoV proteins to romaine lettuce. All data demonstrated that the surface-displayed HuNoV proteins could be utilized in a new and simple system for investigation of the interaction between the HuNoVs and their candidate ligands.
... This criticism has been intensified due to the recent outbreaks linked with Kimchi consumption; the first one took place in 2012 and was caused by enterotoxigenic Escherichia coli O169 while the second occurred in 2013 and was caused by norovirus GI.4 (Cho et al., 2014;Park et al., 2015). From a microbiological point of view, it has been widely accepted that lactic acid fermented foods are not a common vehicle of foodborne pathogens due to the antagonistic effect of the LAB; however, several studies as well as these outbreaks, have highlighted that such generalizations may be uncertain for reasons referring to both the raw materials and the adaptability of the pathogens (Burnett et al., 2000;Frank, 2000, 2001;Beuchat, 2002;Klerks et al., 2007;Kroupitski et al., 2009Kroupitski et al., , 2011Mitra et al., 2009;Critzer and Doyle, 2010;Warriner and Namvar, 2010). Therefore, the ability of pathogenic bacteria to survive and proliferate during kimchi fermentation or storage has been studied to some extent. ...
Different types of fermented foods such as chongkukjang, doenjang, ganjang, gochujang, and kimchi are plentifully available and widely consumed in north eastern Asian countries including Korea. Among them, kimchi is one of the most popular Korean traditional food. It is prepared by fermenting the baechu cabbage together with other vegetables and lactic acid bacteria (LAB) with functional potential. Many types of ingredients are added to kimchi to enhance its taste, flavor, nutritional value, texture etc. A number of bacteria are involved in the fermentation of kimchi, but LAB are the dominant species in the fermentation process. The addition of other sub ingredients and formation of different by-products during fermentation eventually leads to eradication of putrefactive and pathogenic bacteria, and also increase the functionalities, nutritional and nutraceutical potential of kimchi. Kimchi possesses anti-inflammatory, antibacterial, antioxidant, anticancer, antiobesity, probiotic properties, cholesterol reduction, and antiaging properties. In the present review an attempt has been made to review the different types of fermented foods found in the Korean peninsula with detailed scientific research regarding preparation, processing, structure of the microecosystem, and health benefits of kimchi.
... Higher numbers of E. coli O157:H7 (Sakai) were recovered from the roots compared to the phyllosphere since the rhizosphere is a more hospitable environment protected from desiccation and UV irradiation that occur above ground, and has been reported to support substantially higher levels of other human pathogens (Brandl et al., 2004;Kroupitski et al., 2011). In general, higher levels of persistence were observed on the abaxial surfaces of leaves, which is also likely due to differences in UV irradiation and desiccation (Brandl, 2006). ...
Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonisation. As such, we compared the ability of the VTEC strain, E. coli O157:H7 ‘Sakai’, to colonise the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum) and prickly lettuce (L. serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analysed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonisation. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 hour) transcriptional response to extracts as well as longer-term (10 days) plant colonisation or persistence. We show that propagation temperature (37 versus 18 oC) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilisation. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress or translational stalling. A large proportion of differentially regulated genes are uncharacterised (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.
... As reported by Taban and Halkman (2011), both in Europe and in the United States, recent data revealed an increased association of leafy vegetables, especially lettuce and spinach and their RTE salads, with foodborne disease outbreaks. In addition, the edible part of the plant can be contaminated during cutting or pressing of fresh fruits to obtain the juice (Goldberg et al., 2011;Kroupitski et al., 2011;Doyle and Buchanan, 2012). Table 8.2 presents some data on the incidence of Salmonella in leafy vegetables. ...
Changes in dietary habits have pushed the inclusion of numerous ready-to-eat products in the market. Among them, minimally processed vegetables are of great importance because of their convenience, freshness, and nutritional values. The increasing demand for these foods has led to a rise in the association of these foods with disease outbreaks. The concerns with the occurrence of these outbreaks are related to the associated morbidity and mortality rates. Producers also suffer from losses in crops, food products, and corporate image. Leafy vegetables can carry potentially pathogenic microorganisms that may impact public health. This chapter focuses on the aspects related to the occurrence and importance of Salmonella spp. and Listeria monocytogenes in leafy vegetables. The main outbreaks associated with the consumption of leafy vegetables contaminated by these pathogens and the measures to prevent their transmission are discussed.
... The space between an oil gland and surrounding leaf surface provides shelter and good attachment for a microorganism. Microorganisms, especially a mixed culture of S. Typhimurium, tend to extensively colonize the wall of stomata in lettuce (Jahid, Han, Srey, Ha, 2014Ha, , 2015Kroupitski, Pinto, Belausov, & Sela, 2011). However, in sweet basil the microorganisms preferred to attach around the oil glands rather than on the stomata wall as they would in lettuce. ...
... More accurately, these concerns referred mainly to the ability of human pathogens to adapt and survive in extreme micro-environments, such as brined pickles, for extended periods of time (Castanie-Cornet et al. 1999, Tetteh et al. 2001, Mazzotta 2001, Kanellou et al. 2013. These factors, combined with the diversity of the ecological niches that are formed due to substantial diff erences in surface morphology, tissue composition and metabolic activities (Beuchat 2002, Klerks et al. 2007, Mitra et al. 2009, Burnett et al. 2000 of the plant parts used as raw materials along with their interaction with the pathogenic microorganisms (Brandl 2006, Raybaudi-Massilia et al. 2009, Critzer and Doyle 2010, Cooley et al. 2006, Kroupitski et al. 2011) renders at least uncertain any generalization schemes regarding the safety of these products. ...
... Three-dimensional volumetric rendering of CLSM data using specialized software allows viewing of images from varying angles, providing a greater understanding of the sample's spatial arrangement. Many studies have involved the CLSM as a principal tool to study the localization and internalization of bacterial 67,84,116,117 and protozoon 133 foodborne pathogens in fruits and vegetables. Other diverse applications in food microbiology have included determinations of physiologically active foodborne pathogens in foods, 39,44 removal of foodborne pathogens from fresh produce, 202 distribution of bacterial populations in dairy products 16,102,130 and other foods, 15 permeabilization and lysis of starter cultures in Gouda cheese, 38 in vitro attachment of foodborne pathogens to meat proteins, 211 spatial and temporal determinations of foodborne pathogens in biofilms, 164 and comparison and growth and determination of fungal hyphae. ...
Plants may harbor the human pathogen Salmonella enterica. Interactions between S. enterica and different plant species have been studied in individual reports. However, disparities arising from the distinct experimental conditions may render a meaningful comparison very difficult. This study explored interaction patterns between different S. enterica strains including serovars Typhimurium 14028s and LT2 and serovar Senftenberg, and different plants (Arabidopsis, lettuce, and tomato) in one approach. Better persistence of S. enterica serovar Typhimurium strains was observed in all tested plants, whereas the resulting symptoms varied depending on plant species. Genes encoding pathogenesis-related proteins were upregulated in plants inoculated with Salmonella. Furthermore, transcriptome of tomato indicated dynamic responses to Salmonella, with strong and specific responses already 24 h after inoculation. By comparing with publicly accessible Arabidopsis and lettuce transcriptome results generated in a similar manner, constants and variables were displayed. Plants responded to Salmonella with metabolic and physiological adjustments, albeit with variability in reprogrammed orthologues. At the same time, Salmonella adapted to plant leaf-mimicking media with changes in biosynthesis of cellular components and adjusted metabolism. This study provides insights into the Salmonella-plant interaction, allowing for a direct comparison of responses and adaptations in both organisms.
Low‐energy X‐rays can be used to reduce the number of pathogenic microorganisms in fresh produce, but the efficacy of this process against internalized bacteria in leafy greens has not yet been reported. The leaves of iceberg lettuce were cut into pieces and subjected to vacuum perfusion to force the foodborne pathogen cells into the intercellular spaces within the leaves. Sodium hypochlorite (200–400 ppm) washes were not effective in inactivating internalized bacterial cells from lettuce leaves. In contrast, treatment with 1.5 kGy low‐energy X‐rays reduced Escherichia coli O157: H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes levels by 6.89, 4.48, and 3.22 log CFU/g, respectively. Additionally, the maximum dose of X‐rays did not adversely affect the color or texture of lettuce. These results suggest that low‐energy X‐ray treatment can be used to control internalized and surface‐adhering pathogens in leafy vegetables without affecting product quality.
Ready-to-eat fruit and vegetables are a convenient source of nutrients and fibre for consumers, and are generally safe to eat, but are vulnerable to contamination with human enteric bacterial pathogens. Over the last decade, Salmonella spp., pathogenic Escherichia coli, and Listeria monocytogenes have been linked to most of the bacterial outbreaks of foodborne illness associated with fresh produce. The origins of these outbreaks have been traced to multiple sources of contamination from pre-harvest (soil, seeds, irrigation water, domestic and wild animal faecal matter) or post-harvest operations (storage, preparation and packaging). These pathogens have developed multiple processes for successful attachment, survival and colonization conferring them the ability to adapt to multiple environments. However, these processes differ across bacterial strains from the same species, and across different plant species or cultivars. In a competitive environment, additional risk factors are the plant microbiome phyllosphere and the plant responses; both factors directly modulate the survival of the pathogens on the leaf’s surface. Understanding the mechanisms involved in bacterial attachment to, colonization of, and proliferation, on fresh produce and the role of the plant in resisting bacterial contamination is
therefore crucial to reducing future outbreaks.
Human infections by gastrointestinal bacterial pathogens are commonly associated with the consumption of contaminated food of animal origin (e.g., chicken, fish, eggs) or contaminated water. However, further contamination sources must be considered since number of Salmonella enterica infections associated with the consumption of food of non-animal origin (e.g., vegetables, fruits, nuts) are increasing. This gives raise to interest in understanding the interaction of S. enterica with leafy produce, such as various salads. Especially adhesion as initial step of contamination of salad by S. enterica deserves further investigation. Here we introduce methods to analyse Salmonella adhesion to various salads that provide insights into bacterial factors involved in Salmonella colonization of plants.
In a previous study, comparing the internalization of S. enterica serovar Typhimurium in various leaves by confocal microscopy, we have demonstrated that the pathogen failed to internalize tomato leaves. Numerous reasons may account for these findings, yet one such factor might be the methodology employed to quantify leaf internalization. To this end, we have systematically studied leaf localization of a Green-fluorescent protein-labeled Salmonella strain in tomato, lettuce, and Arabidopsis leaves by surface sterilization and enumeration of the surviving bacteria, side by side, with confocal microscopy observations. Leaf sterilization was performed using either sodium hypochlorite, silver nitrate, or ethanol for 1 to 7min. The level of internalization varied according to the type of disinfectant used for surface sterilization and the treatment time. Treatment of tomato leaves with 70% ethanol for up to 7min suggested possible internalization of Salmonella, while confocal microscopy showed no internalization. In the case of in lettuce and Arabidopsis leaves, both the plate-count technique and confocal microscopy demonstrated considerable Salmonella internalization thought different sterilization conditions resulted in variations in the internalization levels. Our findings highlighted the dependency of the internalization results on the specific disinfection protocol used to determine bacterial localization. The results underscore the importance of confocal microscopy in validating a particular surface sterilization protocol whenever a new pair of bacterial strain and plant cultivar is studied.
The produce industry commonly uses chlorine to wash vegetables during post-harvest practices. However, chlorine has disadvantages because it is not sustainable to the environment, is not user-friendly, is corrosive to equipment, and loses efficacy in the presence of organic matter. Hence, alternatives to wash sanitizers are required to better meet the needs of the industry. Essential oil-based antimicrobial microemulsions in the wash water were evaluated for their efficacy against antibiotic-resistant Salmonella enterica serovar Newport and spoilage bacterium Lactobacillus casei on Iceberg lettuce. The microemulsions that were assessed included oregano oil, lemongrass oil, and cinnamon oil along with a plant-based emulsifier for improved solubility of the oil in water. Iceberg lettuce (10 g) was washed thoroughly and inoculated with Lactobacilli casei (6.0 log colony-forming units (CFU)/g) or Salmonella Newport (6.0 log CFU/g). The Iceberg leaves were separately treated with 0.1%, 0.3%, or 0.5% of the microemulsions, 50 ppm chlorine, and 3% hydrogen peroxide, stored at 4 °C, observed, and analyzed for surviving populations of both bacteria on days 0, 3, 7, 10, 14, 21, and 28. The efficacies of the antimicrobials were dependent on concentration and storage-time. The use of microemulsions resulted in a 2.3-4.37 log CFU/g reduction in the Salmonella population at various time points during days 0-28. They were also effective against Lactobacillus, resulting in 0.11-4.25 log CFU/g reduction during storage at days 0-28. The data on visual observation of treated leaves summarized in the tables and shown as figures indicated that the 0.1% oregano oil microemulsion had the best visual appeal in Iceberg leaves inoculated with S. Newport and the 0.5% lemongrass oil microemulsion showed improvement in reduced browning of the Iceberg leaves inoculated with Lactobacillus casei. This study demonstrates the potential of essential oil microemulsions to inactivate foodborne pathogenic and spoilage bacteria on Iceberg lettuce, thus providing effective produce decontamination strategies.
Concerted efforts of scientists to enhance food productivity and nutrition are important to mitigate hunger and malnutrition. At the same time, it is essential that crops be microbiologically safe. This chapter addresses various microbiological issues associated with several types of agricultural crops, including fruits, vegetables, nuts, and grains. One common attribute of such crops is that they are primarily grown in open fields, where sources of microbial contamination may be difficult to control. Hence, the microbiological profile of a crop will vary by the plant type, the region in which it is grown, and the management and processing practices applied to it. To systematically address this topic, this chapter first provides a short description of the food groups discussed. After reviewing some of the major microbial groups associated with each crop group, the chapter addresses (i) quality and safety repercussions associated with microbial contamination of these food groups; (ii) sources of contamination; (iii) detection of contamination; (iv) interventions available to reduce microbial contamination, including physical, chemical, biological, and hurdle treatments; and (v) quantitative microbial risk assessment. Over the past decade, considerable research has been directed toward understanding and mitigating spoilage and pathogen risks associated with these crops, which should lead to extended shelf lives and reduced risk of illnesses associated with these plant‐based foods.
Salmonella enterica is one of the most common pathogens associated with produce outbreaks worldwide; nonetheless, the mechanisms uncovering their interaction with plants are elusive. Previous reports demonstrate that S. enterica ser. Typhimurium (STm), similar to the phytopathogen Pseudomonas syringae pv. tomato (Pst) DC3000, triggers a transient stomatal closure suggesting its ability to overcome this plant defense and colonize the leaf apoplast. In order to discover new molecular players that function in the stomatal re-opening by STm and Pst DC3000, we performed an Arabidopsis mutant screening using thermal imaging. Further stomatal bioassay confirmed that the mutant plants exo70h4-3, sce1-3, bbe8, stp1, and lsu2 have smaller stomatal aperture widths than the wild type Col-0 in response to STm 14028 s. The mutants bbe8, stp1, and lsu2 have impaired stomatal movement in response to Pst DC3000. These findings indicate that EXO70H4 and SCE1 are involved in bacterial-specific responses, while BBE8, STP1 and LSU2 may be required for stomatal response to a broad range of bacteria. The identification of new molecular components of the guard cell movement induced by bacteria will enable a better understanding of the initial stages of plant colonization and facilitate targeted prevention of leaf contamination with harmful pathogens.
Outbreaks associated with fresh-cut leafy greens continue to occur despite efforts to implement horticultural practices that minimize introduction of enteric pathogens to the crop. The experimental trials in this study were designed to examine the efficacy of an acetic acid (AA)- and chitosan-based spray treatment, applied 1 day prior to harvest, for reducing the prevalence of Escherichia coli O157:H7 (O157) and Salmonella in field-grown leafy greens contaminated at levels detectable only through enrichment culture. Responses to the treatment solution were variable and depended on the type of leafy green (leafy lettuce, spinach, or cabbage), cultivar, pathogen, and AA concentration (0.3 to 0.7%). No significant differences in E. coli O157 prevalence were found for untreated and treated cabbage heads and spinach plants (P > 0.05). In contrast, treatment significantly affected Salmonella on 'Bravo F1' green cabbage and '7-Green' spinach (P < 0.05), with odds ratios of 2.2 and 3.3 for finding the pathogen on untreated versus treated greens, respectively. Salmonella was also 7.1 times more likely to be found on an untreated lettuce plant than on a lettuce plant sprayed with a 0.7% AA treatment solution (95% confidence interval [CI], 4.1 to 12.2; P < 0.0001). In studies addressing the efficacy of chitosan (0.1 or 0.3%), this chemical failed to reduce the prevalence of either pathogen on lettuce (P > 0.05). Similarly, spraying with 0.3% AA did not affect the prevalence of Salmonella on lettuce plants (P > 0.05); however, treatment solutions with 0.4% AA reduced the likelihood of detecting Salmonella in treated versus untreated plants by 6.6 times (95% CI, 2.1 to 20.9; P = 0.0007). After the lettuce was harvested and hand washed, consumers failed to distinguish either visually or organoleptically between untreated lettuce and lettuce sprayed with an acetic acid solution (P > 0.05). These results indicate that acetic acid could be used to reduce the microbiological risk of preharvest leafy greens.
Plant pathogenic Pseudomonas species produce effectors, toxins, and cyclic‐lipopeptides to infect various host plants. In spite of many studies to understand the underlying mechanisms of virulence in Pseudomonas spp., the function of genes in the srf operon including srfC, which was formerly known as HopL1, remains undetermined. To investigate the roles of srf genes in the virulence of the bacterial pathogen, we knocked out each srfA, srfB, srfC, and srfD gene from the srf cluster of Pseudomonas cichorii JBC1. When the knocked‐out mutants were infected into tomato seedlings by flood‐inoculation, disease incidence was suppressed only in srfC‐defective mutants (∆srfC) compared to wild‐type (WT). Interestingly, when the ∆srfC strain was directly inoculated into the apoplast of tomato leaves by vacuum infiltration, disease developed similar to that of WT. In addition, the ∆srfC strain showed defective swarming motility and biofilm formation, and the attachment of ∆srfC cells on the leaf surface was significantly reduced compared to WT. Furthermore, droplets of the ∆srfC culture supernatant did not collapse on a hydrophobic surface while the WT collapsed. In summary, our results indicated that SrfC plays important roles in swarming motility, biofilm formation, and attachment/colonization on host surfaces, which is beneficial for the pathogen's dispersion and entry into host tissues and consequently contributes to the disease development. This study elucidates for the first time the functional role of srfC in P. cichorii virulence. The results will broaden our understanding in plant and bacterial pathogen interactions.
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Bacterial contamination of fresh produce is a growing concern in food industry. Pathogenic bacteria can attach to and colonize the surfaces of fresh produce and cause disease outbreaks among consumers. Surface properties of both bacteria and produce affect bacterial contamination; however, the effects of produce roughness, topography, and hydrophobicity on bacterial retention are still poorly understood. In this work, we used spherical polystyrene colloids as bacterial surrogates to investigate colloid retention on and removal (by rinsing) from fresh produce surfaces including tomato, orange, apple, lettuce, spinach, and cantaloupe, and from surrogate produce surface Sharklet (a micro-patterned polymer). All investigated surfaces were characterized in terms of surface roughness and hydrophobicity (including contact angle and water retention area measurements). The results showed that there was no single parameter that dominated colloid retention on fresh produce, yet strong connection was found between colloid retention and water retention and distribution on all the surfaces investigated except apple. Rinsing was generally not efficient in removing colloids from produce surfaces, which suggests the need to modify current cleaning procedures and to develop novel contamination prevention strategies. This work offers a physicochemical approach to a food safety problem and improves understanding of mechanisms leading to produce contamination.
To prevent contamination of fresh produce with enteric pathogens, more insight into mechanisms that may influence the association of these pathogens with fresh produce is needed. In this study, Escherichia coli O157:H7 and Salmonella were chosen as model pathogens, and fresh cut iceberg lettuce was chosen as a model fresh produce type. The morphological structure of iceberg lettuce leaves (stomatal density and length of cell margins per leaf area) was quantified by means of leaf peels and light microscopy of leaves at different stages of development (outer, middle, and inner leaves of the crop) on both leaf sides (abaxial and adxial) and in three leaf regions (top, center, and bottom). The morphology of the top region of the leaves was distinctly different from that of the center and base, with a significantly higher stomatal density (up to five times more stomata), different cell shape, and longer cell margins (two to three times longer). Morphological differences between the same regions of the leaves at different stages of development were smaller or nonsignificant. An attachment assay with two attenuated E. coli O157:H7 strains (84-24h11-GFP and BRMSID 188 GFP) and two Salmonella strains (serovars Thompson and Typhimurium) was performed on different regions of the middle leaves. Our results confirmed earlier reports that these pathogens have a higher affinity for the base of the lettuce leaf than the top. Differences of up to 2.12 log CFU/g were seen (E. coli O157:H7 86-24h11GFP). Intermediate attachment occurred in the central region. The higher incidence of preferential bacterial attachment sites such as stomata and cell margins or grooves could not explain the differences observed in the association of the tested pathogens with different regions of iceberg lettuce leaves.
It is becoming clear that human enteric pathogens, like Salmonella, can efficiently colonize vegetative and reproductive organs of plants. Even though the bacterium's ability to proliferate within plant tissues has been linked to outbreaks of salmonellosis, little is known about regulatory and physiological adaptations of Salmonella, or other human pathogens, to their persistence in plants. A screen of Salmonella deletion mutants in tomatoes identified rcsA and rcsB genes as those under positive selection. In tomato fruits, populations of Salmonella rcsB mutants were as much as 100-fold lower than those of the wild type. In the follow-up experiments, competitive fitness of rcsA and rcsB mutants was strongly reduced in tomatoes. Bioinformatics predictions identified a putative Salmonella RcsAB binding box (TTMGGAWWAABCTYA) and revealed an extensive putative RcsAB regulon, of which many members were differentially fit within tomatoes. This article is protected by copyright. All rights reserved.
The development of rapid detection technologies in parallel with understanding production practices will markedly improve the microbiological safety of leafy vegetables. The objective of this study was on the application of phage‐based magnetoelastic (ME) biosensors and optimization of selected surface blocking agents for a rapid detection for Salmonella Typhimurium on Romaine lettuce. Initially, Romaine lettuce hearts surface was examined for structural differences followed by attachment and distribution of S. Typhimurium using scanning electron microscopy (SEM). Furthermore, selected surface blocking agents were compared and optimized for the application of phage‐based ME biosensors surface. A total of 156 observations, ranging from 1 to 8 logs CFU/ml, and 18 frequency measurements were recorded and analyzed. Both measurement (with E2 phage) and control (without E2 phage) sensors were blocked with selected blocking agents to prevent nonspecific binding. Statistical analysis using ANOVA revealed that there was a significant difference between controls and measurements (P < 0.001). The comparison showed that 5 × 10² CFU/mL and higher concentrations of S. Typhimurium were detected on Romaine lettuce surface, and PEG performed better as a blocking agent of choice. Understanding the complexity of the bacteria‐plant surface interaction and improved detection protocol will provide a solid foundation to design feasible control strategies.
Practical Applications
Salmonella species are generally associated with food products of animal origin and recently leafy green vegetables have been considered as a major public health concern and source of the bacteria. There is still a need to explore swift and reliable detection protocol, which could be easily applied to leafy vegetables from the farm to consumer. In this study, we have demonstrated the rapid detection for Salmonella Typhimurium on Romaine lettuce using phage‐based protocol. Furthermore, better surface blocking agent was optimized for the protocol. Further optimization of phage‐based detection approach could be instrumental to develop better Salmonella detection approach that can be adopted and used from farms to the consumer level.
Spray washing is a common sanitizing method for the fresh produce industry. The purpose of this research was to investigate the antimicrobial effect of spraying slightly acidic electrolyzed water (SAEW) and a combination of ozonated water with ultraviolet (UV) in reducing Escherichia coli O157:H7 on romaine and iceberg lettuces. Both romaine and iceberg lettuces were spot inoculated with 100 μL of a 3 strain mixture of E. coli O157:H7 to achieve an inoculum of 6 log CFU/g on lettuce. A strong antimicrobial effect was observed for the UV-ozonated water combination, which reduced the population of E. coli by 5 log CFU/g of E. coli O157:H7 on both lettuces. SAEW achieved about 5 log CFU/g reductions in the bacterial counts on romaine lettuce. However, less than 2.5 log CFU/g in the population of E. coli O157:H7 was reduced on iceberg lettuce. The difference may be due to bacteria aggregation near and within stomata for iceberg lettuce but not for romaine lettuce. The UV light treatment may stimulate the opening of the stomata for the UV-ozonated water treatment and hence achieve better bacterial inactivation than the SAEW treatment for iceberg lettuce. Our results demonstrated that the combined treatment of SAEW and UV-ozonated water in the spray washing process could more effectively reduce E. coli O157:H7 on lettuce, which in turn may help reduce incidences of E. coli O157:H7 outbreaks.
To improve the microbiological safety of wild vegetables after harvest, Aster scaber and Cirsium setidens Nakai were treated with combinations of 50 ppm aqueous chlorine dioxide (ClO2)/0.5% citric acid or fumaric acid, and 50 ppm ClO2/0.5% fumaric acid/blanching at 90°C for 2 min. Combined treatment of 50 ppm ClO2 and 0.5% citric acid reduced populations of total aerobic bacteria, yeast, and molds in Aster scaber and Cirsium setidens Nakai by 2.80∼3.64 and 2.02∼2.67 log CFU/g, respectively, compared to those of the control. Combined treatment of 50 ppm ClO2 and 0.5% fumaric acid reduced total aerobic bacteria, yeast and molds populations by 3.62∼3.82 and 2.47∼3.02 log CFU/g, respectively. Based on the results, combined treatment of ClO2 and fumaric acid was more effective in controlling microorganisms in the wild vegetables than either ClO2 or citric acid. In addition, combined treatment of ClO2/fumaric acid/blanching reduced the populations of total aerobic bacteria by 4.59∼5.12 log CFU/g, and populations of yeast and molds were not detected by treatment. These results suggest that combined treatment of ClO2/fumaric acid/blanching is the most effective method for improving microbiological safety of wild vegetables after harvest. © 2016, Korean Society of Food Science and Nutrition. All rights reserved.
Understanding the infection mechanisms of pathogens will lead to better management of the associated diseases. The flagella of these pathogens play significant roles not only in bacterial motility, but also in virulence. In the present study, two genes involved in flagella construction, fliJ and fliI of Pseudomonas cichorii, were analyzed. The results revealed that these genes are vital for flagella formation and play significant roles not only in motility, but also in virulence. When we inoculated host plants with fliI- and fliJ-defective mutants (ΔfliJ and ΔfliI) through the dipping method, the degree of disease severity caused by both mutants was significantly reduced compared to those of the wild-type. However, the virulence of ΔfliI was stronger than that of ΔfliJ. Electron microscope observation and swarming and leaf attachment assays indicated a reduced number of flagella in ΔfliI, but not complete absence, because of the presence of another copy of fliI. Furthermore, a vacuum infiltration assay revealed that flagella are indispensable in the pre- and post-penetration stages for complete virulence. Overall, we created semi-defective (ΔfliI) and completely defective (ΔfliJ) mutants and elucidated the fact that flagella play significant roles in virulence of the pathogen at different stages of the infection process.
With mounting evidence indicating an increase in produce (fruits, vegetables and ready-to-eat food) contamination along the food continuum, food safety authorities and the scientific community are examining the best options to prevent contamination of food (pre- and post-harvest) before it reaches the consumer, as well as devising intervention strategies to combat and contain any inadvertent post-harvest food contamination.
Outbreak information linking fresh tomato fruit to illnesses is reviewed in this chapter. While tomato fruit appear to support substantial proliferation of certain serovars of Salmonella enterica, detection of this pathogen in tomato plants prior to harvest is rare, and reports of Salmonella existence in tomato fruit still attached to field-grown plants are virtually non-existent. The bacterium is sensitive to UV and can be outcompeted by the native phytomicrobiota, which may explain its absence in field-grown crops. However, the persistence of certain serovars in fields and ponds of certain production areas is noted. Together with evidence of bacteria becoming internalized in tomato fruit during crop development likely through natural apertures, the presence of S. enterica in and around production fields suggests that an unusual weather event could lead to Salmonella contamination of fruit prior to harvest. The bacterium appears physiologically adaptive toward proliferation in tomato fruit. Once inside tomatoes, Salmonella is capable of sensing the availability of nutrients and physiological state of the fruit and differentially regulates specific genes. However, because Salmonella is an efficient nutrient scavenger, removal of multiple metabolic and regulatory genes was required to reduce its fitness within the fruit. Plants do not appear to recognize human enterics as pathogens, and their defenses treat them as endophytes.
Outbreaks associated with fresh produce have been traced to farms in several cases. Potential sources of contamination in preharvest environments have been identified, and minimizing their input is needed. In addition, understanding the fate of enteric pathogens introduced to soil and plant systems is essential to providing safe guidelines on when crops may be planted and harvested. Moisture availability and temperature are key abiotic factors affecting pathogen survival. Indigenous soil and epiphytic bacteria, however, also appear to play an important role in a pathogen's fate and thus future survival studies should routinely monitor the types and levels present. Internalization of enteric pathogens through lateral root junctions or through leaf stomata has been documented but generally requires high exposure concentrations. Plant defenses, whether basal or activated by the invading enteric pathogen, appear to inactivate internalized populations as persistence has not been observed, but this subject deserves further investigation.
Fruits and vegetables are beneficial for human health. A major contribution to this comes from the microorganisms associated with them. However, fruits and vegetables may cause food poisoning, when contaminated with human pathogens and eaten raw. In our review, based on a literature survey, we show insights into the entry of Escherichia coli or Salmonella enterica into the fruit and vegetable environment via diverse routes and the complex interactions between various biotic and abiotic factors, which influence the survival of human pathogens in soil and on plants. In particular, mobile genetic elements acquired through horizontal gene transfer contribute to the diversity of pathogenic strains and to their adaptation to extra-intestinal habitats. For a successful establishment of human pathogens on plants, the attachment, internalization and the response of the plant are important. Mainly the genetic equipment of the human pathogen, the plant, and its microbiome determine thecomplex interaction between human pathogens and plants. However, also a multitude of environmental factors plays an important role. The availability of nutrients, for example, is fundamental for the survival of human pathogens and they have to compete for them with the indigenous plant and soil microorganisms. Recommendations for the practice require an improved understanding of the ecology of human pathogens in soil and on plants but also along the production chain. The development and use of sensitive and specific detection tools is essential and needs to consider the diversification of human pathogens through horizontal gene transfer as well as the problem of their non-cultivability under environmental stress conditions.
Lettuce is a vegetable that is always consumed raw. The number and types of microorganisms that may be found on the lettuce leaves depend on growing, harvesting, and particularly on handling practices, like proper washing and preparing lettuce before eating. The aim of this study was to investigate the effect that washing under running water has on the microbiological status of lettuce leaves purchased from the local market place in Banja Luka. Smears were taken from the obverse and reverse sides of lettuce leaves, and the total count of aerobic mesophilic bacteria, yeasts and molds was determined as well as the presence of sulphite-reducing clostridia, the coagulase positive staphylococci, Salmonella species, and Escherichia coli. The analysis showed that the washing reduced the total count of aerobic mesophilic bacteria by approximately 10 times, and the total count of yeasts and molds by approximately 8 times. Of the lettuce samples examined, 66,67% contained sulphite-reducing clostridia and 13,33% Salmonella species, before the washing. The number of samples with sulphite-reducing clostridia was reduced by five times after the washing, but the number of samples contaminated with Salmonella species remained the same.
The number of outbreaks of foodborne illness arising from the consumption of fresh and fresh-cut produce has risen over the last two decades. As a result of outbreaks occurring since the mid-1990s, the fate of foodborne pathogens in agricultural environments has been studied in detail. Plants do not normally harbor enteric pathogens; however, zoonotic bacteria may transfer from other sources in the field such as soil, manure, compost, irrigation water, insects, and wild or domestic animals. Until recently, it was thought that enteric pathogens survive poorly in the harsh environment encountered on plant surfaces or in the plant tissues, but recent research has shown this not to be the case, and the intimate interactions between enteric pathogens and plant tissue have begun to be scrutinized. This chapter will discuss the attachment of foodborne pathogens to plant tissue and the mechanisms the pathogens employ to persist on/in the plants. It will focus on bacterial systems, evolved in attachment and invasion to the host or in suppression of the immune response in the host, helping the pathogens to survive on/in the plants. Although a variety of microorganisms (bacteria, viruses, parasites) have been implicated in outbreaks arising from produce, this chapter will focus primarily on Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes because of the frequency of outbreaks associated with these pathogens and the depth to which they have been studied.
ABSTRACf This review focuses on the role of two distinct fitness strategies in the growth, survival, and epidemiology of foliar bacterial pathogens. A tolerance strategy requires the ability to tolerate direct exposure to environmental stresses on leaf surfaces, including UV radiation and low water availability. An avoidance strategy requires the ability to seek and/or exploit sites that are protected from these stresses, including endophytic sites. The ability to employ an avoidance strategy and grow endophytically may directly influence the potential for pathogenesis, since endophytic populations, not epiphytic populations, are likely responsible for disease induction. Furthermore, exchange between these two populations is probably crucial to the epidemiology of foliar pathogens. While foliar pathogens can grow and survive in both exposed and internal sites, indicating that they can employ both fitness strategies, the poor internal growth of most saprophytes suggests that saprophytes depend primarily on a strategy of tolerance. This difference between pathogens and saprophytes has important implications for predicting the population dynamics of leaf-associ ated bacterial species and for selecting effective biological control agents.
Contaminated salad leaves have emerged as important vehicles for the transmission of enteric pathogens to humans. A recent outbreak of Salmonella enterica serovar Senftenberg (S. Senftenberg) in the United Kingdom has been traced to the consumption of contaminated basil. Using the outbreak strain of S. Senftenberg, we found that it binds to basil, lettuce, rocket and spinach leaves showing a pattern of diffuse adhesion. Flagella were seen linking S. Senftenberg to the leaf epidermis, and the deletion of fliC (encoding phase-1 flagella) resulted in a significantly reduced level of adhesion. In contrast, although flagella linking S. enterica serovar Typhimurium to the basil leaf epidermis were widespread, deletion of fliC did not affect leaf attachment levels. These results implicate the role of flagella in Salmonella leaf attachment and suggest that different Salmonella serovars use strain-specific mechanisms to attach to salad leaves.Keywords: Salmonella enterica, flagella, salad leaves
Human pathogens can contaminate leafy produce in the field by various routes. We hypothesized that interactions between Escherichia coli O157:H7 and spinach are influenced by the route of introduction and the leaf microenvironment. E. coli O157:H7 labeled with green fluorescent protein was dropped onto spinach leaf surfaces, simulating bacteria-laden raindrops or sprinkler irrigation, and survived on the phylloplane for at least 14 days, with increasing titers and areas of colonization over time. The same strains placed into the rhizosphere by soil infiltration remained detectable on very few plants and in low numbers (10(2) to 10(6) CFU/g fresh tissue) that decreased over time. Stem puncture inoculations, simulating natural wounding, rarely resulted in colonization or multiplication. Bacteria forced into the leaf interior survived for at least 14 days in intercellular spaces but did not translocate or multiply. Three spinach cultivars with different leaf surface morphologies were compared for colonization by E. coli O157:H7 introduced by leaf drop or soil drench. After 2 weeks, cv. Bordeaux hosted very few bacteria. More bacteria were seen on cv. Space and were dispersed over an area of up to 0.3 mm2. The highest bacterial numbers were observed on cv. Tyee but were dispersed only up to 0.15 mm2, suggesting that cv. Tyee may provide protected niches or more nutrients or may promote stronger bacterial adherence. These findings suggest that the spinach phylloplane is a supportive niche for E. coli O157:H7, but no conclusive evidence was found for natural entry into the plant interior. The results are relevant for interventions aimed at minimizing produce contamination by human pathogens.
Outbreaks of salmonellosis related to consumption of fresh produce have raised interest in Salmonella-plant interactions leading to plant colonization. Incubation of gfp-tagged Salmonella enterica with iceberg lettuce leaves in the light resulted in aggregation of bacteria near open stomata and invasion into the inner
leaf tissue. In contrast, incubation in the dark resulted in a scattered attachment pattern and very poor stomatal internalization.
Forcing stomatal opening in the dark by fusicoccin had no significant effect on Salmonella internalization. These results imply that the pathogen is attracted to nutrients produced de novo by photosynthetically active
cells. Indeed, mutations affecting Salmonella motility and chemotaxis significantly inhibited bacterial internalization. These findings suggest a mechanistic account for
entry of Salmonella into the plant's apoplast and imply that either Salmonella antigens are not well recognized by the stoma-based innate immunity or that this pathogen has evolved means to evade it.
Internalization of leaves may provide a partial explanation for the failure of sanitizers to efficiently eradicate food-borne
pathogens in leafy greens.
There is an increased concern that plants might be more important as a carrier for human enteric pathogens like E. coli O157:H7 and Salmonella enterica serovars than previously thought. This review summarizes the knowledge available on the ecology of E. coli O157:H7 and Salmonella enterica in the primary production chain of leafy green vegetables (in particular lettuce), including manure, manure-amended soil, and crop. Based on the available literature, suggestions are made for the control of these pathogens. The suggested approach of oligotrophication of agro-ecosystems fits in the wider approach to lower environmental emissions of nutrients from manure application and to enhance the suppression against plant pathogens.
Confocal scanning laser microscopy was used to observe the location of Escherichia coli O157:H7 on and within lettuce leaves. Sections of leaves (ca. 0.5 by 0.5 cm) were inoculated by submersion in a suspension of E. coli O157:H7 (ca. 10(7) to 10(8) CFU/ml) overnight at 7 degrees C. Fluorescein isothiocyanate-labeled antibody was used to visualize the attached bacteria. E. coli O157:H7 was found attached to the surface, trichomes, stomata, and cut edges. Three-dimensional volume reconstruction of interior portions of leaves showed that E. coli O157:H7 was entrapped 20 to 100 microm below the surface in stomata and cut edges. Agar plate culturing and microscopic observation indicated that E. coli O157:H7 preferentially attached to cut edges, as opposed to the intact leaf surface. Dual staining with fluorescein isothiocyanate-labeled antibody and propidium iodide was used to determine viability of cells on artificially contaminated lettuce leaves after treatment with 20 mg/liter chlorine solution for 5 min. Many live cells were found in stomata and on cut edges following chlorine treatment. E. coli O157:H7 did not preferentially adhere to biofilm produced by Pseudomonas fluorescens on the leaf surface. In contrast to E. coli O157:H7, Pseudomonas adhered to and grew mainly on the intact leaf surface rather than on the cut edges.
Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.
Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria. The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively. E. coli O157:H7 retained pEGFP during frozen storage at -80 degrees C. The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells. A low percentage of EGFP-expressing cells was nonviable. The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration. The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured. The application of EGFP as a marker for E. coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy. EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types. The numbers of E. coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato. E. coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces. EGFP can serve as a marker to characterize E. coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.
Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E. coli O157: H7 for 24+/-1 h at 4 degrees C. Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degrees C. Viability of E. coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue. Most E. coli O157:H7 cells (68.3+/-16.2%) that had penetrated 30 to 40 microm from the damaged tissue surface remained viable after chlorine treatment. Cells on the surface survived least (25.2+/-15.8% survival), while cells that penetrated 0 to 10 microm from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/-13.5 and 45.6+/-9.7% survival, respectively). Viability was associated with the depth at which E. coli O157:H7 cells were in the stomata. Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome. These results demonstrate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.
Leaf surfaces provide the ecologically relevant landscapes to those organisms that encounter or colonize the leaf surface. Leaf surface topography directly affects microhabitat availability for colonizing microbes, microhabitat quality and acceptability for insects, and the efficacy of agricultural spray applications. Prior detailed mechanistic studies that examined particular fungi-plant and pollinator-plant interactions have demonstrated the importance of plant surface topography or roughness in determining the outcome of the interactions. Until now, however, it has not been possible to measure accurately the topography--i.e., the three-dimensional structure--of such leaf surfaces or to record precise changes in patterns of leaf surface elevation over time. Using contact mode atomic force microscopy, we measured three-dimensional coordinates of upper leaf surfaces of Vaccinium macrocarpon (cranberry), a perennial plant, on leaves of two age classes. We then produced topographic maps of these leaf surfaces, which revealed striking differences between age classes of leaves: old leaves have much rougher surfaces than those of young leaves. Atomic force microscope measurements were analyzed by lag (1) autocorrelation estimates of leaf surfaces by age class. We suggest that the changes in topography result from removal of epicuticular lipids and that the changes in leaf surface topography influence phylloplane ecology. Visualizing and mapping leaf surfaces permit detailed investigations into leaf surface-mediated phenomena, improving our understanding of phylloplane interactions.
The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning
microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of
plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22°C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable
bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30°C, after inoculation, indicating
that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance
of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization
by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates
on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.
Numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks have been associated with contaminated sprouts. We examined how S. enterica serovars, E. coli serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on and adhere to alfalfa sprouts. Growth on and
adherence to sprouts were not significantly different among different serovars of S. enterica, but all S. enterica serovars grew on and adhered to alfalfa sprouts significantly better than E. coli O157:H7. E. coli O157:H7 was essentially rinsed from alfalfa sprouts with repeated washing steps, while 1 to 2 log CFU of S. enterica remained attached per sprout. S. enterica Newport adhered to 3-day-old sprouts as well as Pantoea agglomerans and 10-fold more than Pseudomonas putida and Rahnella aquatilis, whereas the growth rates of all four strains throughout seed sprouting were similar. S. enterica Newport and plant-associated bacteria adhered 10- to 1,000-fold more than E. coli O157:H7; however, three of four other E. coli serotypes, isolated from cabbage roots exposed to sewage water following a spill, adhered to sprouts better than E. coli O157:H7 and as well as the Pseudomonas and Rahnella strains. Therefore, attachment to alfalfa sprouts among E. coli serotypes is variable, and nonpathogenic strains of E. coli to be used as surrogates for the study of pathogenic E. coli may be difficult to identify and should be selected carefully, with knowledge of the biology being examined.
Fresh produce is an important part of a healthy diet. During the last three decades, the number of outbreaks caused by foodborne pathogens associated with fresh produce consumption reported to the Centers for Disease Control and Prevention has increased. To identify trends, we analyzed data for 1973 through 1997 from the Foodborne Outbreak Surveillance System. We defined a produce-associated outbreak as the occurrence of two or more cases of the same illness in which epidemiologic investigation implicated the same uncooked fruit, vegetable, salad, or juice. A total of 190 produce-associated outbreaks were reported, associated with 16,058 illnesses, 598 hospitalizations, and eight deaths. Produce-associated outbreaks accounted for an increasing proportion of all reported foodborne outbreaks with a known food item, rising from 0.7% in the 1970s to 6% in the 1990s. Among produce-associated outbreaks, the food items most frequently implicated included salad, lettuce, juice, melon, sprouts, and berries. Among 103 (54%) produce-associated outbreaks with a known pathogen, 62 (60%) were caused by bacterial pathogens, of which 30 (48%) were caused by Salmonella. During the study period, Cyclospora and Escherichia coli O157:H7 were newly recognized as causes of foodborne illness. Foodborne outbreaks associated with fresh produce in the United States have increased in absolute numbers and as a proportion of all reported foodborne outbreaks. Fruit and vegetables are major components of a healthy diet, but eating fresh uncooked produce is not risk free. Further efforts are needed to better understand the complex interactions between microbes and produce and the mechanisms by which contamination occurs from farm to table.
Numerous Salmonella enterica food-borne illness outbreaks have been associated with contaminated vegetables, in particular sprouted seeds, and the incidence
of reported contamination has steadily risen. In order to understand the physiology of S. enterica serovar Newport on plants, a screen was developed to identify transposon mutants that were defective in attachment to alfalfa
sprouts. Twenty independent mutants from a pool of 6,000 were selected for reduced adherence to alfalfa sprouts. Sixty-five
percentage of these mutants had insertions in uncharacterized genes. Among the characterized genes were strains with insertions
in the intergenic region between agfB, the surface-exposed aggregative fimbria (curli) nucleator, and agfD, a transcriptional regulator of the LuxR superfamily, and rpoS, the stationary-phase sigma factor. Both AgfD and RpoS have been reported to regulate curli and cellulose production and
RpoS regulates other adhesins such as pili. The intergenic and rpoS mutants were reduced in initial attachment to alfalfa sprouts by 1 log unit compared to the wild type. Mutations of agfA, curli subunit, and agfB in S. enterica serovar Enteritidis differentially affected attachment to plant tissue. The agfA mutation was not reduced in ability to attach to or colonize alfalfa sprouts, whereas the agfB mutation was reduced. Thus, agfB alone can play a role in attachment of S. enterica to plant tissue. These results reveal that S. enterica genes important for virulence in animal systems are also required for colonization of plants, a secondary host that can serve
as a vector of S. enterica from animal to animal.
In this study, we assessed various leaf structural and chemical features as possible predictors of the size of the phyllosphere bacterial population in the Mediterranean environment. We examined eight perennial species, naturally occurring and coexisting in the same area, in Halkidiki (northern Greece). They are Arbutus unedo, Quercus coccifera, Pistacia lentiscus, and Myrtus communis (evergreen sclerophyllous species), Lavandula stoechas and Cistus incanus (drought semi-deciduous species), and Calamintha nepeta and Melissa officinalis (non-woody perennial species). M. communis, L. stoechas, C. nepeta, and M. officinalis produce essential oil in substantial quantities. We sampled summer leaves from these species and (1) estimated the size of the bacterial population of their phyllosphere, (2) estimated the concentration of different leaf constituents, and (3) studied leaf morphological and anatomical features and expressed them in a quantitative way. The aromatic plants are on average more highly colonized than the other species, whereas the non-woody perennials are more highly colonized than the woody species. The population size of epiphytic bacteria is positively correlated with glandular and non-glandular trichome densities, and with water and phosphorus contents; it is negatively correlated with total phenolics content and the thickness of the leaf, of the mesophyll, and of the abaxial epidermis. No correlation was found with the density of stomata, the nitrogen, and the soluble sugar contents. By regression tree analysis, we found that the leaf-microbe system can be effectively described by three leaf attributes with leaf water content being the primary explanatory attribute. Leaves with water content >73% are the most highly colonized. For leaves with water content <73%, the phosphorus content, with a critical value of 1.34 mg g(-1) d.w., is the next explanatory leaf attribute, followed by the thickness of the adaxial epidermis. Leaves higher in phosphorus (>1.34 mg g(-1) d.w.) are more colonized, and leaves with the adaxial epidermis thicker than 20.77 microm are the least colonized. Although these critical attributes and values hold true only within the Mediterranean ecosystem studied and the range of observations taken, they are important because they provide a hypothesis to be tested in other Mediterranean ecosystems and other biomes. Such comparative studies may give insight as to the general properties governing the leaf-microbe system.
Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease
in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types
of surfaces. Several E. coli O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether
mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to
plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in
vitro. The genes tested included those encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA, yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD, and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci) into an E. coli K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these
genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the ompA gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest
that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three
types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable
for binding to plant surfaces was ompA.
Diarrheagenic Escherichia coli were able to bind to plant surfaces, including alfalfa sprouts and open seed coats, and tomato and Arabidopsis thaliana seedlings incubated in water. The characteristics of the binding differed with the bacterial strain examined. Laboratory K12 strains of E. coli failed to show significant binding to any of the plant surfaces examined, suggesting that some of the genes present and expressed in pathogenic strains and absent or unexpressed in K12 strains may be required for binding to plants. When a plasmid carrying the mlrA gene (a positive regulator of curli biosynthesis) or a plasmid carrying the operons that encode the synthesis of curli (csgA-G) was introduced into K12 strains, the bacteria acquired the ability to bind to sprouts. CsgA mutants of an avian pathogenic E. coli and an O157:H7 strain showed no reduction in their ability to bind to sprouts. Thus, the production of curli appears to be sufficient to allow K12 strains to bind, but curli are not necessary for the binding of pathogenic strains, suggesting that pathogenic strains may have more than one mechanism for binding to plant surfaces.
The effect of the disinfectant sodium hypochlorite (NaClO), with or without mild heat (50 degrees C) and fumaric acid, on native bacteria and the foodborne pathogens Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium DT104 attached to iceberg lettuce leaves was examined. The retail lettuce examined consistently harbored 6 to 7 log CFU/g of native bacteria throughout the study period. Inner leaves supported 1 to 2 log CFU/g fewer bacteria than outer leaves. About 70% of the native bacterial flora was removed by washing five times with 0.85% NaCl. S. aureus, E. coli, and Salmonella allowed to attach to lettuce leaves for 5 min were more easily removed by washing than when allowed to attach for 1 h or 2 days, with more S. aureus being removed than E. coli or Salmonella Typhimurium. An increase of time for attachment of pathogens from 5 min to 2 days leads to decreased efficiency of the washing and sanitizing treatment. Treatment with fumaric acid (50 mM for 10 min at room temperature) was the most effective, although it caused browning of the lettuce, with up to a 2-log reduction observed. The combination of 200 ppm of sodium hypochlorite and mild heat treatment at 50 degrees C for 1 min reduced the pathogen populations by 94 to 98% (1.2- to 1.7-log reduction) without increasing browning.
Total, fluorescent, and pectolytic epiphytic bacterial population sizes were quantified on leaves of different age groups of broad-leaved endive during field cultivation from leaf emergence until harvest. Greater bacterial population densities (log(inf10) CFU per square centimeter) were observed on outer leaves than on inner leaves of the plants throughout the growing season. These differences were statistically significant for total bacterial populations at all sampling times and were often significant for fluorescent and pectolytic bacterial populations. At harvest, a linear gradient of decreasing densities of epiphytic bacteria from outer (older) to inner (younger) leaves of the head was significant. Leaf age influenced the frequency distribution and variability of bacterial population sizes associated with leaves of broad-leaved endive. Total bacterial population sizes were greater at leaf emergence for leaves emerging during the second half of the cultivation period than for leaves emerging earlier. The size of fluorescent and pectolytic bacterial populations on newly emerged leaves increased throughout the season as plants aged. To assess the importance of plant age on bacterial immigration at leaf emergence, bacterial densities were quantified on leaves emerging simultaneously on plants of different ages. In two of the three experiments, greater bacterial population sizes were observed on leaves emerging on younger plants. This indicates that factors other than an increase in concentration of airborne bacteria can lead to increases in population sizes at leaf emergence as plants age in the field. Results of leaf pruning experiments suggested that adjacent leaves may act as a barrier for immigration of fluorescent bacteria on newly emerged leaves. Survival of an inoculated strain of Pseudomonas fluorescens on newly emerged leaves generally did not vary with the age of plants. However, these effects were not consistent among experiments, suggesting that interactions among micro- and macroenvironmental conditions, physiological condition of leaves, and accessibility of leaves to airborne bacteria are important in controlling epiphytic bacterial population sizes.
Numerous salmonellosis outbreaks have been associated with vegetables, in particular sprouted seed. Thin aggregative fimbriae (Tafi), a component of the extracellular matrix responsible for multicellular behavior, are important for Salmonella enterica attachment and colonization of plants. Here, we demonstrate that the other surface polymers composing the extracellular matrix, cellulose, and O-antigen capsule also play a role in colonization of plants. Mutations in bacterial cellulose synthesis (bcsA) and O-antigen capsule assembly and translocation (yihO) reduced the ability to attach to and colonize alfalfa sprouts. A colanic acid mutant was unaffected in plant attachment or colonization. Tafi, cellulose synthesis, and O-antigen capsule, all of which contribute to attachment and colonization of plants, are regulated by AgfD, suggesting that AgfD is a key regulator for survival outside of hosts of Salmonella spp. The cellulose biosynthesis regulator adrA mutant was not affected in the ability to attach to or colonize plants; however, promoter probe assays revealed expression by cells attached to alfalfa sprouts. Furthermore, quantitative reverse-transcriptase polymerase chain reaction revealed differential expression of agfD and adrA between planktonic and plant-attached cells. In addition, there was no correlation among mutants between biofilm formation in culture and attachment to plants. Outside of animal hosts, S. enterica appears to rely on an arsenal of adhesins to persist on plants, which can act as vectors and perpetuate public health concerns.
The availability of knowledge of the route of infection and critical plant and microbe factors influencing the colonization efficiency of plants by human pathogenic bacteria is essential for the design of preventive strategies to maintain safe food. This research describes the differential interaction of human pathogenic Salmonella enterica with commercially available lettuce cultivars. The prevalence and degree of endophytic colonization of axenically grown lettuce by the S. enterica serovars revealed a significant serovar-cultivar interaction for the degree of colonization (S. enterica CFUs per g leaf), but not for the prevalence. The evaluated S. enterica serovars were each able to colonize soil-grown lettuce epiphytically, but only S. enterica serovar Dublin was able to colonize the plants also endophytically. The number of S. enterica CFU per g of lettuce was negatively correlated to the species richness of the surface sterilized lettuce cultivars. A negative trend was observed for cultivars Cancan and Nelly, but not for cultivar Tamburo. Chemotaxis experiments revealed that S. enterica serovars actively move toward root exudates of lettuce cultivar Tamburo. Subsequent micro-array analysis identified genes of S. enterica serovar Typhimurium that were activated by the root exudates of cultivar Tamburo. A sugar-like carbon source was correlated with chemotaxis, while also pathogenicity-related genes were induced in presence of the root exudates. The latter revealed that S. enterica is conditioned for host cell attachment during chemotaxis by these root exudates. Finally, a tentative route of infection is described that includes plant-microbe factors, herewith enabling further design of preventive strategies.
This review focuses on the role of two distinct fitness strategies in the growth, survival, and epidemiology of foliar bacterial pathogens. A tolerance strategy requires the ability to tolerate direct exposure to environmental stresses on leaf surfaces, including UV radiation and low water availability. An avoidance strategy requires the ability to seek and/or exploit sites that are protected from these stresses, including endophytic sites. The ability to employ an avoidance strategy and grow endophytically may directly influence the potential for pathogenesis, since endophytic populations, not epiphytic populations, are likely responsible for disease induction. Furthermore, exchange between these two populations is probably crucial to the epidemiology of foliar pathogens. While foliar pathogens can grow and survive in both exposed and internal sites, indicating that they can employ both fitness strategies, the poor internal growth of most saprophytes suggests that saprophytes depend primarily on a strategy of tolerance. This difference between pathogens and saprophytes has important implications for predicting the population dynamics of leaf-associated bacterial species and for selecting effective biological control agents.
Outbreaks of Escherichia coli O157:H7 infections have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence
that this enteric pathogen can multiply on the leaves of romaine lettuce plants. The increases in population size of E. coli O157:H7 in the phyllosphere of young lettuce plants ranged from 16- to 100-fold under conditions of warm temperature and
the presence of free water on the leaves and varied significantly with leaf age. The population size was consistently ca.
10-fold higher on the young (inner) leaves than on the middle leaves. The growth rates of Salmonella enterica and of the natural bacterial microflora were similarly leaf age dependent. Both enteric pathogens also achieved higher population
sizes on young leaves than on middle leaves harvested from mature lettuce heads, suggesting that leaf age affects preharvest
as well as postharvest colonization. Elemental analysis of the exudates collected from the surfaces of leaves of different
ages revealed that young-leaf exudates were 2.9 and 1.5 times richer in total nitrogen and carbon, respectively, than middle-leaf
exudates. This trend mirrored the nitrogen and carbon content of the leaf tissue. Application of ammonium nitrate, but not
glucose, to middle leaves enhanced the growth of E. coli O157:H7 significantly, suggesting that low nitrogen limits its growth on these leaves. Our results indicate that leaf age
and nitrogen content contribute to shaping the bacterial communities of preharvest and postharvest lettuce and that young
lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7.
U.S. salmonellosis outbreaks have occurred following consumption of tomato and cantaloupe but not lettuce. We report differential
contamination among agricultural seedlings by Salmonella enterica via soil. Members of the family Brassicaceae had a higher incidence of outbreak than carrot, lettuce, and tomato. Once they
were contaminated, phyllosphere populations were similar, except for tomato. Contamination differences exist among tomato
cultivars.
To quantify the variability and dynamics of bacterial populations on field-grown broad-leaved endive (Cichorium endivia cv. Samy) destined for ready-to-use processing, plants were sown at three dates in 1990 for cultivation during the autumn season in southern France. Densities of populations of total, fluorescent pseudomonad and pectolytic bacteria were determined for entire seedlings in the greenhouse and for outer and inner leaves of plants in the field throughout the season until harvest. On seedlings from the greenhouse, bacteria were always detected but fluorescent and pectolytic bacteria were only sporadically detected and generally composed less than 0.1% of the total population. In the field there were significant differences in total, fluorescent and pectolytic bacterial populations on outer and inner leaves, among different plants of the same age, and among inner leaves sampled at the three different dates of harvest. the variability observed suggests that it is reasonable to pursue development of cultural or industrial sorting methods to reduce the microbial load of endive destined for ready-to-use processing.
Aims: To investigate the interactions of Salmonella enterica with abiotic and plant surfaces and their effect on the tolerance of the pathogen to various stressors.
Methods and Results: Salmonella strains were tested for their ability to form biofilm in various growth media using a polystyrene plate model. Strong biofilm producers were found to attach better to intact Romaine lettuce leaf tissue compared to weak producers. Confocal microscopy and viable count studies revealed preferential attachment of Salmonella to cut-regions of the leaf after 2 h at 25°C, but not for 18 h at 4°C. Storage of intact lettuce pieces contaminated with Salmonella for 9 days at 4°C resulted only in small changes in population size. Exposure of lettuce-associated Salmonella cells to acidic conditions (pH 3·0) revealed increased tolerance of the attached vs planktonic bacteria.
Conclusions: Biofilm formation on polystyrene may provide a suitable model to predict the initial interaction of Salmonella with cut Romaine lettuce leaves. Association of the pathogen with lettuce leaves facilitates its persistence during storage and enhances its acid tolerance.
Significance and Impact of the Study: Understanding the interactions between foodborne pathogens and lettuce might be useful in developing new approaches to prevent fresh produce-associated outbreaks.
Storage of partially processed lettuce resulted in an increase in microbial population, pH, and change in package atmosphere composition (increasing CO2 and decreasing O2). Microbial populations varied in size with variations in processing and packaging. Typical initial log10 counts per g were: bacteria 3.4–5.1, yeasts 2.5–3.2; molds were infrequent. Commercial packaging inhibited bacterial growth and retarded browning. Although Gram-negative bacteria were numerically dominant, a large yeast population was also found. Species in the genera Pseudomonas, Erwinia, and Serratia were the most frequently isolated bacteria. Cryptococcus, Pichia, Torulaspora and Trichosporon spp. were the most common yeasts.
Traditional antimicrobials have been extensively used for many years. However, consumers are currently demanding wholesome, fresh-like, and safe foods without addition of chemically synthesized preservatives. The application of novel natural antimicrobials to assure safety of fresh-cut fruits and unpasteurized juices while preventing quality loss is a promising alternative. The effectiveness of these natural substances added to fruit derivatives has been studied by different researchers. Antimicrobials of animal (lactoperoxidase, lysozyme, and chitosan), plant (essential oils, aldehydes, esters, herbs, and spices), and microbial origin (nisin) can be used to effectively reduce pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices. Nevertheless, the use of these compounds at a commercial level is still limited due to several factors such as impact on sensory attributes or, in some cases, regulatory issues concerning their use. Therefore, extensive research on the effects of each antimicrobial on food sensory characteristics is still needed so that antimicrobial substances of natural origin can be regarded as feasible alternatives to synthetic ones.
Epiphytic microbial communities of terrestrial plants are non uniformly distributed in space and in time on leaf surfaces.
The size and composition of microbial populations vary under the influence of biotic and abiotic factors related to the micro-organisms
themselves (traits conferring epiphytic fitness, nutritional resource utilisation, abilities to compete for space, resistance
or production of toxic compounds), to the host (its genotype, the age and the position of the leaves), and to the environmental
conditions (micro- and macro-climate, activity of vectors and pathogens, application of pesticides and other chemicals). Moreover,
the microbial population dynamics results from four processes -immigration, emigration, multiplication and death of cells
as discussed in a previous chapter (Lindow, this volume) — which are under the influence of the previously mentioned factors.
The recent recognition of fresh fruits and vegetables as major vehicles of foodborne illness has led to increased research on mechanisms by which enteric pathogens contaminate and persist on and in this non-host environment. Interactions between foodborne pathogens and plants as well among the naturally occurring microbial communities contribute to endophytic and epiphytic colonization. Scientific findings are just beginning to elucidate the mechanisms that contribute to colonization of produce. This review addresses current knowledge as well as future research needed to increase our understanding of the microbial ecology of enteric pathogens on fruits and vegetables.
Through recent advances in our understanding of microbial:plant interactions it is becoming apparent that human pathogens, principally, Escherichia coli O157:H7 and Salmonella are adapted to survive in the plant environment. The aforementioned pathogens have surface epitopes that can bind to plant structures such as stomata to aid attachment. In addition, Salmonella is attracted and able to metabolize nutrients contained within the apoplastic fluid of plants. The question of internalization into the inner tissue of plants remains inconclusive largely because of the problems encountered in detecting low pathogen levels. Nevertheless, once internalized human pathogens can trigger and potentially evade plant defenses that are typically induced by phytopathogens. Although more research in this area is required, the hypothesis that human pathogens have adapted to the plant environment as part of their natural lifecycle appears to be supported.
The surface of plant leaves - the phyllosphere - is home to many microbes. A 'community proteogenomics' approach offers a fresh look at what it takes to survive and thrive in this unique habitat.
Foodborne illness outbreaks linked to fresh produce are becoming more frequent and widespread. High impact outbreaks, such as that associated with spinach contaminated with Escherichia coli O157:H7, resulted in almost 200 cases of foodborne illness across North America and >$300 m market losses. Over the last decade there has been intensive research into gaining an understanding on the interactions of human pathogens with plants and how microbiological safety of fresh produce can be improved. The following review will provide an update on the food safety issues linked to fresh produce. An overview of recent foodborne illness outbreaks linked to fresh produce. The types of human pathogens encountered will be described and how they can be transferred from their normal animal or human host to fresh produce. The interaction of human pathogens with growing plants will be discussed, in addition to novel intervention methods to enhance the microbiological safety of fresh produce.
Foodborne Salmonella spp. is a leading cause of foodborne illness in the United States each year. Traditionally, most cases of salmonellosis were thought to originate from meat and poultry products. However, an increasing number of salmonellosis outbreaks are occurring as a result of contaminated produce. Several produce items specifically have been identified in outbreaks, and the ability of Salmonella to attach or internalize into vegetables and fruits may be factors that make these produce items more likely to be sources of Salmonella. In addition, environmental factors including contaminated water sources used to irrigate and wash produce crops have been implicated in a large number of outbreaks. Salmonella is carried by both domesticated and wild animals and can contaminate freshwater by direct or indirect contact. In some cases, direct contact of produce or seeds with contaminated manure or animal wastes can lead to contaminated crops. This review examines outbreaks of Salmonella due to contaminated produce, the potential sources of Salmonella, and possible control measures to prevent contamination of produce.
Recent outbreaks of gastroenteritis linked to the consumption of fresh produce raise questions about the mechanisms by which human pathogens colonize plants and persist within marketable produce. Neither Salmonella nor Escherichia coli appear to produce enzymes that degrade plant cell walls, therefore it is not yet certain how these bacteria enter plant tissues and spread within them. Similar to plant-associated bacteria, enterics use cellulose and aggregative fimbriae for their attachment to plant surfaces. Salmonella can be an effective plant endophyte, even though it is capable of triggering plant defenses. Plant-associated microbiota contributes to the fitness and translocation of these human pathogens within plant hosts, although interactions and mechanisms of communication between plant-associated microbiota and enteric pathogens are not yet characterized.
1. The predominant organisms isolated from the outer wrapper leaves of freshly harvested white cabbages were: bacteria, yeasts, Alternaria spp., Aureobasidium pullulans, Botrytis cinerea, Cladosporium spp. and Penicillium spp.
2. Few qualitative or quantitative changes were seen in the leaf surface flora during storage at 2°C for up to 33 weeks.
3. Numbers of bacteria, particularly fluorescent and pectolytic pseudomonads, were considerably higher on cabbages drenched with fungicide or water than on corresponding undrenched cabbages.
One-hundred and twenty samples of lettuce and 89 samples of fennel purchased from five retail outlets in the city of Bari (Italy) from October 1973 through September 1975 were examined for viable aerobic bacteria (AB), total coliforms (TC), fecal coliforms (FC), fecal streptococci (FS), and salmonellae. Comparative tests indicated that the results of bacteriological analysis of wash water from either vegetable by a membrane filter technique compared favorably with those of conventional cultural examination of the vegetable tissue for the purpose of providing an indication of the bacteriological quality of the samples. Using the membrane filter technique, 2-year average counts of 6.59 X 10(7) for AB, 5.95 X 10(4) for TC, 6.13 X 10(3) for FC and 2.24 X 10(3) for FS/100 g (fresh weight) were obtained with lettuce; with fennel, the corresponding figures were 2.32 X 10(6) for AB, 7.82 X 10(4) for TC, 7.8 X 10(4) for FC, and 3.15 X 10(3) for FS. Indicator bacteria were present in all samples examined. In addition, 68.3% of the lettuce and 71.9% of the fennel samples yielded one or more of the following Salmonella serotypes: S. schottmuelleri, S. typhimurium, S. thompson, S. dublin, S. typhi, and S. anatum.
A study was undertaken to determine the survival patterns of Salmonella montevideo G4639 on and in tomatoes during storage and the efficacy of chlorine treatment on inactivation of the pathogen. The population of S. montevideo on the surfaces of inoculated tomatoes stored at 10 degrees C did not change significantly (P < 0.05) throughout an 18-day storage period. Significant increases in population occurred within 7 days and within 1 day when tomatoes were stored at 20 and 30 degrees C, respectively. A significantly higher number of cells was taken up by the core tissue of tomatoes tempered at 25 degrees C when the tomatoes were dipped in a suspension at 10 degrees C compared with the number taken up when the tomatoes were dipped in cell suspensions tempered at 25 or 37 degrees C. Populations remained constant throughout subsequent storage for 8 days at 10 degrees C, regardless of the temperature differential between tomatoes and the dip suspension. Storage of tomatoes at 20 degrees C, however, resulted in significant increases in populations of S. montevideo. Populations of the pathogen on the surfaces and in the core tissues of tomatoes were significantly reduced by dipping for 2 min in a solution containing 60 or 110 ppm (60 or 110 micrograms/ml) chlorine, respectively; however, treatment in solution containing 320 ppm chlorine did not result in complete inactivation. Populations of S. montevideo remained unchanged in chopped tomatoes stored at 5 degrees C for 216 h (9 days) but increased significantly after storage for 96 or 22 h at 20 or 30 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Outbreaks of human infections associated with consumption of raw fruits and vegetables have occurred with increased frequency during the past decade. Factors contributing to this increase may include changes in agronomic and processing practices, an increase in per capita consumption of raw or minimally processed fruits and vegetables, increased international trade and distribution, and an increase in the number of immuno-compromised consumers. A general lack of efficacy of sanitizers in removing or killing pathogens on raw fruits and vegetables has been attributed, in part, to their inaccessibility to locations within structures and tissues that may harbor pathogens. Understanding the ecology of pathogens and naturally occurring microorganisms is essential before interventions for elimination or control of growth can be devised.
The continuous rise in the number of outbreaks of foodborne illness linked to fresh fruit and vegetables challenges the notion that enteric pathogens are defined mostly by their ability to colonize the intestinal habitat. This review describes the epidemiology of produce-associated outbreaks of foodborne disease and presents recently acquired knowledge about the behavior of enteric pathogens on plants, with an emphasis on Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. The growth and survival of enteric pathogens on plants are discussed in the light of knowledge and concepts in plant microbial ecology, including epiphytic fitness, the physicochemical nature of plant surfaces, biofilm formation, and microbe-microbe and plant-microbe interactions. Information regarding the various stresses that affect the survival of enteric pathogens and the molecular events that underlie their interactions in the plant environment provides a good foundation for assessing their role in the infectious dose of the pathogens when contaminated fresh produce is the vehicle of illness.
Consumption of fruit and vegetable products is commonly viewed as a potential risk factor for infection with enteropathogens such as Salmonella and Escherichia coli O157, with recent outbreaks linked to lettuce, spinach and tomatoes. Routes of contamination are varied and include application of organic wastes to agricultural land as fertilizer, contamination of waters used for irrigation with faecal material, direct contamination by livestock, wild animals and birds and postharvest issues such as worker hygiene. The ability of pathogens to survive in the field environment has been well studied, leading to the implementation of guidelines such as the Safe Sludge Matrix, which aim to limit the likelihood of viable pathogens remaining at point-of-sale. The behaviour of enteropathogens in the phyllosphere is a growing field of research, and it is suggested that inclusion in phyllosphere biofilms or internalization within the plant augments the survival. Improved knowledge of plant-microbe interactions and the interaction between epiphytic and immigrant micro-organisms on the leaf surface will lead to novel methods to limit enteropathogen survival in the phyllosphere.
Microbiology of the phyllosphere Mapping leaf surface landscapes
- S E Lindow
- M T Brandl
- W L Mechaber
- D B Marshall
- R A Mechaber
- R T Jobe
- F S Chew
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Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere Microbial ecology of foodborne pathogens associated with produce Bacteriological quality assessment of fresh marketed lettuce and fennel
- M T Brandl
- R E Mandrell
- F J Critzer
- M P Doyle
- G L Ercolani
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