Changes in spinach phylloepiphytic bacteria communities following minimal processing and refrigerated storage described using pyrosequencing of 16S rRNA amplicons

The phyllosphere is colonized by a wide variety of bacteria and fungi; it harbors epiphytes, as well as plant-pathogenic bacteria and even human pathogens. However, little is known about how the bacterial community composition on leafy greens develops over time. The bacterial community of the leafy-green phyllosphere obtained from two plantings of rocket salad (Diplotaxis tenuifolia) and three plantings of lettuce (Lactuca sativa) at two farms in Norway were profiled by an Illumina MiSeq-based approach. We found that the bacterial richness of the L. sativa samples was significantly greater shortly (3 weeks) after planting than at harvest (5 to 7 weeks after planting) for plantings 1 and 3 at both farms. For the second planting, the bacterial diversity remained consistent at the two sites. This suggests that the effect on bacterial colonization of leaves, at least in part must, be seasonally driven rather than driven solely by leaf maturity. The distribution of phyllosphere communities varied between D. tenuifolia and L. sativa at harvest. The variability between these species at the same location suggests that the leaf-dwelling bacteria are not only passive inhabitants but interact with the host, which shapes niches favoring the growth of particular taxa. This work contributes to our understanding of host plant-specific microbial community structures and shows how these communities change throughout plant development.T he phyllosphere is a habitat on the surface of plant leaves colonized by a variety of bacteria, yeasts, and fungi (1). It harbors epiphytes, as well as plant-pathogenic bacteria and even human pathogens. Microbial populations on leaf surfaces are highly influenced by rapid fluctuations in UV radiation, temperature, and humidity and are restricted by limited access to nutrients (1, 2). Resident bacteria on leaves can have neutral, negative, or positive influences on their host plants by serving as pathogens, preventing leaf colonization by pathogens, or acting as growth promoters (3). Traditionally, phyllosphere bacteria have been characterized by using culture-based approaches and much of the work on produce-associated bacteria has focused on a small number of pathogenic species. Culture-based methods will not include bacteria that are not able to grow on standard artificial media or are slow growing. This limits our understanding of the phyllosphere microbial community's ecology, genetics, and physiology (4). Bacterial communities associated with leafy greens have already been described by several culture-independent studies. "First-generation" molecular techniques have been used to describe variation in community structure in the context of leaf surface properties and microbial interactions (5), seasonal variation in the community structure (6), and monitoring of bacterial communities in the food chain (7). The introduction of culture-independent methods, in particular, microbial profiling using high-throughput sequencing to study microorganisms, has revealed more complexity and diversity of the...

Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 91%

mentioning

confidence: 99%

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Bacterial Communities Associated with Surfaces of Leafy Greens: Shift in Composition and Decrease in Richness over Time

Dees

Lysøe

Nordskog

et al. 2015

“…2). Similar to other studies of edible leaf communities (27,37), Pseudomonadales (primarily Pseudomonas) was the most dominant taxon observed on leaf surfaces. While on average they represented 46% of the bacteria on leaves, their proportion on individual leaf samples varied widely, ranging from 4 to 86%.…”

Section: Resultssupporting

confidence: 83%

High-Level Culturability of Epiphytic Bacteria and Frequency of Biosurfactant Producers on Leaves

Burch

Paulina

Sbodio

et al. 2016

To better characterize the bacterial community members capable of biosurfactant production on leaves, we distinguished culturable biosurfactant-producing bacteria from nonproducers and used community sequencing to compare the composition of these distinct cultured populations with that from DNA directly recovered from leaves. Communities on spinach, romaine, and head lettuce leaves were compared with communities from adjacent samples of soil and irrigation source water. Soil communities were poorly described by culturing, with recovery of cultured representatives from only 21% of the prevalent operational taxonomic units (OTUs) (>0.2% reads) identified. The dominant biosurfactant producers cultured from soil included bacilli and pseudomonads. In contrast, the cultured communities from leaves are highly representative of the culture-independent communities, with over 85% of the prevalent OTUs recovered. The dominant taxa of surfactant producers from leaves were pseudomonads as well as members of the infrequently studied genus Chryseobacterium. The proportions of bacteria cultured from head lettuce and romaine leaves that produce biosurfactants were directly correlated with the culture-independent proportion of pseudomonads in a given sample, whereas spinach harbored a wider diversity of biosurfactant producers. A subset of the culturable bacteria in irrigation water also became enriched on romaine leaves that were irrigated overhead. Although our study was designed to identify surfactant producers on plants, we also provide evidence that most bacteria in some habitats, such as agronomic plant surfaces, are culturable, and these communities can be readily investigated and described by more classical culturing methods. IMPORTANCEThe importance of biosurfactant production to the bacteria that live on waxy leaf surfaces as well as their ability to be accurately assessed using culture-based methodologies was determined by interrogating epiphytic populations by both culture-dependent and culture-independent methods. Biosurfactant production was much more frequently observed in cultured communities on leaves than in other nearby habitats, such as soil and water, suggesting that this trait is important to life on a leaf by altering either the leaf itself or the interaction of bacteria with water. While pseudomonads were the most common biosurfactant producers isolated, this habitat also selects for taxa, such as Chryseobacterium, for which this trait was previously unrecognized. The finding that most epiphytic bacterial taxa were culturable validates strategies using more classical culturing methodologies for their study in this habitat. The phyllosphere is recognized to be a selective habitat in which epiphytic bacteria must be able to access limited and spatially heterogeneous nutrient supplies and endure frequent fluctuations in moisture availability on a water-repellent surface (1, 2). Recent culture-independent community analyses have confirmed the apparent selectivity of leaf surfaces, finding that leaf sur...

“…This method of determining the composition of bacterial communities has been applied to surfaces of ready-to-eat vegetables, including spinach (10)(11)(12)(13). Using such an approach to determine the presence of foodborne pathogens in the bacterial community associated with fresh bagged leafy greens may provide the ability to detect pathogens more quickly than current methods permit.…”

mentioning

confidence: 99%

Application of Metagenomic Sequencing to Food Safety: Detection of Shiga Toxin-Producing Escherichia coli on Fresh Bagged Spinach

Leonard

Mammel

Lacher

et al. 2015

109

107

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (ϳ10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection. Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause severe illness such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans (1). Fresh leafy vegetables have been implicated in many foodborne disease outbreaks, including an STEC O157:H7 outbreak associated with consumption of fresh ready-to-eat bagged spinach in 2006 that caused 100 hospitalizations and five deaths (2, 3). In an unrelated outbreak, bagged baby spinach was recalled in 2010 due to STEC O157:H7 contamination (4). The Centers for Disease Control and Prevention reported that between 1973 and 2012, STEC was second only to norovirus in the number of outbreaks associated with leafy vegetables in the United States, of which 46 of the 49 STEC outbreaks were caused by a serogroup O157 strain (2). Although STEC did not cause the greatest number of outbreaks, it did lead to by far the greatest number of hospitalizations for a given microorganism, with 46% of the total hospitalizations due to all leafy-vegetable-associated outbreaks attributed to STEC. Illness due to ingestion of STEC-contaminated leafy greens led to nine deaths, including six from spinach (2). With ...