Dataset Information

Seasonality, shelf life and storage atmosphere are main drivers of the microbiome and E. coli O157:H7 colonization of post-harvest lettuce cultivated in a major production area in California.

ABSTRACT:

Background

Lettuce is linked to recurrent outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections, the seasonality of which remains unresolved. Infections have occurred largely from processed lettuce, which undergoes substantial physiological changes during storage. We investigated the microbiome and STEC O157:H7 (EcO157) colonization of fresh-cut lettuce of two cultivars with long and short shelf life harvested in the spring and fall in California and stored in modified atmosphere packaging (MAP) at cold and warm temperatures.

Results

Inoculated EcO157 declined significantly less on the cold-stored cultivar with short shelf life, while multiplying rapidly at 24 °C independently of cultivar. Metagenomic sequencing of the lettuce microbiome revealed that the pre-storage bacterial community was variable but dominated by species in the Erwiniaceae and Pseudomonadaceae. After cold storage, the microbiome composition differed between cultivars, with a greater relative abundance (RA) of Erwiniaceae and Yersiniaceae on the cultivar with short shelf life. Storage at 24 °C shifted the microbiome to higher RAs of Erwiniaceae and Enterobacteriaceae and lower RA of Pseudomonadaceae compared with 6 °C. Fall harvest followed by lettuce deterioration were identified by recursive partitioning as important factors associated with high EcO157 survival at 6 °C, whereas elevated package CO₂ levels correlated with high EcO157 multiplication at 24 °C. EcO157 population change correlated with the lettuce microbiome during 6 °C storage, with fall microbiomes supporting the greatest EcO157 survival on both cultivars. Fall and spring microbiomes differed before and during storage at both temperatures. High representation of Pantoea agglomerans was a predictor of fall microbiomes, lettuce deterioration, and enhanced EcO157 survival at 6 °C. In contrast, higher RAs of Erwinia persicina, Rahnella aquatilis, and Serratia liquefaciens were biomarkers of spring microbiomes and lower EcO157 survival.

Conclusions

The microbiome of processed MAP lettuce evolves extensively during storage. Under temperature abuse, high CO₂ promotes a lettuce microbiome enriched in taxa with anaerobic capability and EcO157 multiplication. In cold storage, our results strongly support a role for season and lettuce deterioration in EcO157 survival and microbiome composition, suggesting that the physiology and microbiomes of fall- and spring-harvested lettuce may contribute to the seasonality of STEC outbreaks associated with lettuce grown in coastal California.

SUBMITTER: Leonard SR

PROVIDER: S-EPMC8686551 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Plant injury is inherent to the production and processing of fruit and vegetables. The opportunistic colonization of damaged plant tissue by human enteric pathogens may contribute to the occurrence of outbreaks of foodborne illness linked to produce. Escherichia coli O157:H7 (EcO157) responds to physicochemical stresses in cut lettuce and lettuce lysates by upregulation of several stress response pathways. We investigated the tolerance of EcO157 to osmotic stress imposed by the leakage of osmolytes from injured lettuce leaf tissue. LC-MS analysis of bacterial osmoprotectants in lettuce leaf lysates and wound washes indicated an abundant natural pool of choline, but sparse quantities of glycine betaine and proline. Glycine betaine was a more effective osmoprotectant than choline in EcO157 under osmotic stress conditions in vitro. An EcO157 mutant with a deletion of the betTIBA genes, which are required for biosynthesis of glycine betaine from imported choline, achieved population sizes twofold lower than those of the parental strain (P < 0.05) over the first hour of colonization of cut lettuce in modified atmosphere packaging (MAP). The cell concentrations of the betTIBA mutant also were 12-fold lower than those of the parental strain (P < 0.01) when grown in hypertonic lettuce lysate, indicating that lettuce leaf cellular contents provide choline for osmoprotection of EcO157. To demonstrate the utilization of available choline by EcO157 for osmoadaptation in injured leaf tissue, deuterated (D-9) choline was introduced to wound sites in MAP lettuce; LC-MS analysis revealed the conversion of D9-choline to D-9 glycine betaine in the parental strain, but no significant amounts were observed in the betTIBA mutant. The EcO157 ΔbetTIBA-ΔotsBA double mutant, which is additionally deficient in de novo synthesis of the compatible solute trehalose, was significantly less fit than the parental strain after their co-inoculation onto injured lettuce leaves and MAP cut lettuce. However, its competitive fitness followed a different time-dependent trend in MAP lettuce, likely due to differences in O2 content, which modulates betTIBA expression. Our study demonstrates that damaged lettuce leaf tissue does not merely supply EcO157 with substrates for proliferation, but also provides the pathogen with choline for its survival to osmotic stress experienced at the site of injury.

Project description:Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms.IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.

Project description:Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.

Project description:Leafy green vegetables, such as lettuce, have been increasingly implicated in outbreaks of foodborne illnesses due to contamination by Escherichia coli O157:H7. While E. coli can survive in soils, colonize plants, and survive on produce, very little is known about the interaction of E. coli with the roots of growing lettuce plants. In these studies, a combination of microarray analyses and surface enhanced Raman spectroscopy (SERS) were used to gain a comprehensive understanding of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots and compared to E. coli K12 using a hydroponic system (HS) which we have reported in the previous studies. Using microarray, after three days of interaction with lettuce roots, 94 and 109 genes of E. coli O157:H7 were significantly up-regulated and down-regulated at least 1.5 fold, respectively. Only 8 genes were also found in the E. coli K12 up-regulated genes. No genes were found in the down-regulated genes clusters between those two strains. For E. coli O157:H7, forty out of the 94 up-regulated genes (43%) were involved in protein synthesis and were highly repressed compared to 40 out of 193 (23%) E. coli K12 up-regulated genes associated with protein synthesis. The wildtype of E.coli O157:H7 colonized two log CFU per root less compared to E. coli K12. Genes involved in biofilm modulation (bhsA and ybiM) were significantly up-regulated in E. coli O157:H7 and curli production (crl and csgA) were found important for E. coli K12 to attach to lettuce roots in the previous studies. BhsA mutant of E. coli O157:H7 was impaired in the colonization of lettuce roots. The SERS spectra of E. coli K12 and O157 controls (cells without interacting with roots) were very similar. The spectra of E. coli K12 and O157 exposed to the hydroponic system (HS) showed some differences in the nucleic acid, protein, and lipid regions compared with controls. The spectra of E. coli K12 HS cells exhibited significant differences compared to spectra from E. coli O157 HS cells in the RNA and protein regions. The overall band intensity of amide regions declined for E. coli O157 HS cells, while it increased for E. coli K12 HS cells. The intensity of the RNA bands of E. coli K12 HS cells were also found much higher than those of E. coli O157 HS cells. These findings were in agreement to our Microarray data. Our microarray and SERS data showed that E. coli K12 and O157:H7 behavior dramatically differently in colonizing on lettuce roots. Compared to K12, E. coli O157:H7 colonized less efficiently on lettuce roots.

Project description:Escherichia coli O157:H7 (EcO157) infections have been recurrently associated with produce. The physiological state of EcO157 cells surviving the many stresses encountered on plants is poorly understood. EcO157 populations on plants in the field generally follow a biphasic decay in which small subpopulations survive over longer periods of time. We hypothesized that these subpopulations include persister cells, known as cells in a transient dormant state that arise through phenotypic variation in a clonal population. Using three experimental regimes (with growing, stationary at carrying capacity, and decaying populations), we measured the persister cell fractions in culturable EcO157 populations after inoculation onto lettuce plants in the laboratory. The greatest average persister cell fractions on the leaves within each regime were 0.015, 0.095, and 0.221%, respectively. The declining EcO157 populations on plants incubated under dry conditions showed the largest increase in the persister fraction (46.9-fold). Differential equation models were built to describe the average temporal dynamics of EcO157 normal and persister cell populations after inoculation onto plants maintained under low relative humidity, resulting in switch rates from a normal cell to a persister cell of 7.7 × 10-6 to 2.8 × 10-5 h-1 Applying our model equations from the decay regime, we estimated model parameters for four published field trials of EcO157 survival on lettuce and obtained switch rates similar to those obtained in our study. Hence, our model has relevance to the survival of this human pathogen on lettuce plants in the field. Given the low metabolic state of persister cells, which may protect them from sanitization treatments, these cells are important to consider in the microbial decontamination of produce.IMPORTANCE Despite causing outbreaks of foodborne illness linked to lettuce consumption, E. coli O157:H7 (EcO157) declines rapidly when applied onto plants in the field, and few cells survive over prolonged periods of time. We hypothesized that these cells are persisters, which are in a dormant state and which arise naturally in bacterial populations. When lettuce plants were inoculated with EcO157 in the laboratory, the greatest persister fraction in the population was observed during population decline on dry leaf surfaces. Using mathematical modeling, we calculated the switch rate from an EcO157 normal to persister cell on dry lettuce plants based on our laboratory data. The model was applied to published studies in which lettuce was inoculated with EcO157 in the field, and switch rates similar to those obtained in our study were obtained. Our results contribute important new knowledge about the physiology of this virulent pathogen on plants to be considered to enhance produce safety.