More about how the EHEC germ may get transmitted in plant material such as seeds

posted in: Syndicated | 6

It is widely known that pathogenic E. coli germs have ability to survive inside fresh vegetable produce such as spinach, lettuce and sprouts, where they are protected against disinfection. This ability may be a key factor explaining the transmission of the E. coli strain in the dreadful (and still ongoing) German outbreak.

A new article has just come out dissecting how related EHEC E. coli bacteria attach to plant surfaces. They have remarkable complex adhesion abilities that enable the deadly microbes to persist inside plant tissue cavities and on plant surfaces. These consist of various different surface protein hooks that are given names like pili, curli, and flagella.

Although we know that EHEC and EAEC bacteria are a bit different to one another, this new work on EHEC E. coli teaches us a lot about the possible routes of transmission of the German HUS germ– for example through seeds.

A scientific summary on the mechanisms of the EHEC germ attachment to plants is given below. The paper provides access to the latest research on this topic.

 (The term STpEAEC which means Shigatoxin producing EAEC is now being used to describe the German outbreak strain; see previous GMO Pundit post for the latest news about STpEAEC)

Surface structures involved in plant stomata and leaf colonization by Shiga-toxigenic Escherichia coli O157:H7
Zeus Saldaña, Ethel Sánchez, Juan Xicohtencatl-Cortes, Jose Luis Puente and Jorge A. Girón
Fronteers in Microbiology Volune 2 Art 119 Original Research Article published: 27 May 2011 doi: 10.3389/fmicb.2011.00119

Shiga-toxigenic Escherichia coli (STEC) O157:H7 uses a myriad of surface adhesive appendages including pili, flagella, and the type 3 secretion system (T3SS) to adhere to and inflict damage to the human gut mucosa. Consumption of contaminated ground beef, milk, juices, water, or leafy greens has been associated with outbreaks of diarrheal disease in humans due to STEC.

The aim of this study was to investigate which of the known STEC O157:H7 adherence factors mediate colonization of baby spinach leaves and where the bacteria reside within tainted leaves. We found that STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus, hemorrhagic coli type 4 pilus, flagella, and T3SS.

Electron microscopy analysis of tainted leaves revealed STEC bacteria in the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized into stomata more efficiently than without the LEE.

This study highlights a role for pili, flagella, and T3SS in the interaction of STEC with spinach leaves. Colonization of plant stomata and internal tissues may constitute a strategy by which STEC survives in a nutrient-rich microenvironment protected from external foes and may be a potential source for human infection.

Keywords: plant colonization, stomata, STEC, O157:H7, pathogenesis, spinach, T3SS, pili

Hat-tip to Kate Howell.

Further relevant papers
Research
Thomas Breuer and colleagues
Breuer et al, Emerging Infectious Disease, 7:(6) Nov-Dec, 2001.
A multistate outbreak of Escherichia coli O157:H7 infections occurred in the United States in June and July 1997. Two concurrent outbreaks were investigated through independent case-control studies in Michigan and Virginia and by subtyping isolates with pulsed-field gel electrophoresis (PFGE). Isolates from 85 persons were indistinguishable by PFGE. Alfalfa sprouts were the only exposure associated with E. coli O157:H7 infection in both Michigan and Virginia. Seeds used for sprouting were traced back to one common lot harvested in Idaho. New subtyping tools such as PFGE used in this investigation are essential to link isolated infections to a single outbreak….
Trace-Back
Trace-back to the sprouting facility was successful in 29 of 31 instances in which ill persons reported eating alfalfa sprouts. Of 16 successful trace-backs in Michigan, 15 led to one sprouting facility, facility A, in Michigan; one patient could have eaten sprouts from either facility A or facility B in Michigan. All 13 successful trace-backs in Virginia were traced to one sprouting facility in Virginia. During the outbreak period, the Virginia company used only one seed lot. That same seed lot was one of only two lots continuously sprouted by facility A in Michigan from mid-May to the first week of July. Facility B in Michigan sprouted a small number of seeds from this lot on only 2 days; the sprouts from these seeds represented only a fraction of each day’s production. The implicated seed lot was not distributed to any other sprouting company in or outside the United States. That seed lot was 17,000 lbs, of which 6,000 lbs still existed and were immediately removed from distribution.
The implicated seed lot was a blend of five lots from four farms, harvested from 1984 to 1996. The seed processor and the farmers were all located in Idaho. Inspection of the alfalfa fields revealed three possible sources of contamination: cattle manure, water, and deer feces. Manure is not normally applied in alfalfa fields in Idaho; however, cattle feedlots are common in the area. The alfalfa fields of one of the farmers were adjacent to a feedlot. Manure may have leaked or been illegally dumped next to feedlots. In addition, run-off water from neighboring fields, which is collected in furrows and sometimes used to irrigate alfalfa fields, could carry manure to the fields. Three of the four farmers reported at least occasionally seeing deer in their fields. In fact, one had fields next to a wildlife refuge and reported that deer were in the fields every day. Contaminated alfalfa plants, cattle manure, or deer feces could be picked up by the thresher during harvesting and contaminate the seeds. No evidence was found for bacterial contamination at the seed processor.
Ethan B. Solomon, Sima Yaron, and Karl R. Matthews*
Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901
Applied and Environmental Microbiology, January 2002, p. 397-400, Vol. 68, No. 1
The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.
D.L. Jones
DOI: 10.1111/j.1475-2743.1999.tb00069.x
Soil Use and Management
Volume 15, Issue 2, pages 76–83, June 1999
Keywords:
Escherichia coli; pathogens; soil; cattle manure; slurries; transmission; public health; UK
Abstract.  Escherichia coli serotype O157 is a virulent human pathogen the global incidence of which has increased. It has been demonstrated that cattle are the primary reservoir of this pathogen. This has serious implications for the land-based disposal of organic wastes such as cattle manure, cattle slurry and abattoir waste. Further, it also has serious ramifications for the protection of surface and groundwater drinking supplies and public access to pasture land. However, while soil and vegetation can be expected to directly influence the survival of this pathogen, there is a paucity of information concerning the behaviour and survival of E. coli O157 in agricultural environments. It appears that E. coli O157 presently contaminates between 1 to 15% of UK cattle herds, depending on region, and that faecal excretion of the bacterium shows a distinct seasonality which also reflects the incidence of human infections. E. coli O157 can remain viable in soil for greater than 4 months and appears to be a highly resilient pathogen possessing the capability to adapt easily to environmental stresses. While most human cases of E. coli O157 related food poisoning have been associated with the consumption of contaminated meat and dairy products, there is also evidence that human infection has occurred through the ingestion of contaminated soil, fruit and vegetables and drinking water. In this review the potential threat to human health posed by the application of contaminated organic wastes to soil and possible strategies for reducing the amount of pathogen entering the food chain are highlighted.
Ethan B. Solomon, Sima Yaron, and Karl R. Matthews*
Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901
Applied and Environmental Microbiology, January 2002, p. 397-400, Vol. 68, No. 1
The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.
Survival of Escherichia coli O157:H7 in the rhizosphere of maize grown in waste-amended soil.
Williams AP, Avery LM, Killham K, Jones DL.
Source
School of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd, UK. [email protected]
J Appl Microbiol. 2007 Feb;102(2):319-26.
Abstract
AIMS:
To assess whether the persistence of Escherichia coli O157:H7 in soil amended with cattle slurry and ovine stomach content waste is affected by the presence of a maize rhizosphere.
METHODS AND RESULTS:
Cattle slurry and ovine stomach content waste were inoculated with E. coli O157:H7. Wastes were then applied to soil cores with and without established maize plants. The pathogen survived in soil for over 5 weeks, although at significantly greater numbers in soil receiving stomach content waste in comparison to cattle slurry. Persistence of the pathogen in soil was unaffected by the presence of a rhizosphere.
CONCLUSIONS:
Other factors may be more influential in regulating E. coli O157:H7 persistence in waste-amended soil than the presence or absence of a rhizosphere; however, waste type did have significant affect on the survival of E. coli O157:H7 in such soil.
SIGNIFICANCE AND IMPACT OF THE STUDY:
Escherichia coli O157:H7 can be present within animal-derived organic wastes that are routinely spread on land. Introduced measures with regards to such waste disposal may decrease exposure to the organism; however, the persistence of E. coli O157:H7 for considerable periods in waste-amended soil may still pose some risk for both human and animal infection. This study has shown that whilst survival of E. coli O157:H7 in waste-amended soil is not significantly affected by the presence or absence of a maize rhizosphere; it may vary significantly with waste type. This may have implications for land and waste management.
Avery LM, Killham K, Jones DL.
J Appl Microbiol. 2005;98(4):814-22.
PMID: 15752326
Heat and lime-treatment as effective control methods for E. coli O157:H7 in organic wastes.
Avery LM, Williams AP, Killham K, Jones DL.
Bioresour Technol. 2009 May;100(10):2692-8. Epub 2009 Jan 31.
PMID: 19181517
Survival of verocytotoxigenic Escherichia coli O157 in soil, water and on surfaces.
Maule A. Symp Ser Soc Appl Microbiol. 2000;(29):71S-78S. Review.
PMID: 10880181
Solomon EB, Pang HJ, Matthews KR. J Food Prot. 2003 Dec;66(12):2198-202.
PMID: 14672213

Isolation of Salmonella from alfalfa seed and demonstration of impaired growth of heat-injured cells in seed homogenates.
Int J Food Microbiol. 2003 May 15;82(3):245-53.
Liao CH, Fett WF.
Source
US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA. [email protected]
Abstract
Three major foodborne outbreaks of salmonellosis in 1998 and 1999 were linked to the consumption of raw alfalfa sprouts. In this report, an improved method is described for isolation of Salmonella from alfalfa seed lots, which had been implicated in these outbreaks. From each seed lot, eight samples each containing 25 g of seed were tested for the presence of Salmonella by the US FDA Bacteriological Analytical Manual (BAM) procedure and by a modified method applying two successive pre-enrichment steps. Depending on the seed lot, one to four out of eight samples tested positive for Salmonella by the standard procedure and two to seven out of eight samples tested positive by the modified method. Thus, the use of two consecutive pre-enrichment steps led to a higher detection rate than a single pre-enrichment step. This result indirectly suggested that Salmonella cells on contaminated seeds might be injured and failed to fully resuscitate in pre-enrichment broth containing seed components during the first 24 h of incubation. Responses of heat-injured Salmonella cells grown in buffered peptone water (BPW) and in three alfalfa seed homogenates were investigated. For preparation of seed homogenates, 25 g of seeds were homogenized in 200 ml of BPW using a laboratory Stomacher and subsequently held at 37 degrees C for 24 h prior to centrifugation and filtration. While untreated cells grew at about the same rate in BPW and in seed homogenates, heat-injured cells (52 degrees C, 10 min) required approximately 0.5 to 4.0 h longer to resuscitate in seed homogenates than in BPW. This result suggests that the alfalfa seed components or fermented metabolites from native bacteria hinder the repair and growth of heat-injured cells. This study also shows that an additional pre-enrichment step increases the frequency of isolation of Salmonella from naturally contaminated seeds, possibly by alleviating the toxic effect of seed homogenates on repair or growth of injured cells.

Methods for detection of bacteria in seeds

Detection of Salmonella by indicator agar media and PCR as affected by alfalfa seed homogenates and native bacteria
Significance and Impact: The results generated from this study will aid the seed distributors, sprout growers and public health officials to identify and recall the Salmonella-contaminated seed lots to be used for sprout production.
C.-H. Liao and L.M. Shollenberger Letters in Applied Microbiology 2003, 36, 152–15
US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA

Follow David Tribe:
David Tribe is an applied geneticist, teaching graduate/undergrad courses in food science, food safety, biotechnology and microbiology at the University of Melbourne.