What Europeans should have read about pathogenic E. coli in 2007 to have helped avoid the current food safety crisis

posted in: Syndicated | 16
E. coli can survive for a long time on composted soils

Germany is fatally sick. There is a lethal elephant in the room.  This is that elephant:

“Use of animal wastes in fertilization of fresh fruits and vegetable plots significantly increased the risk of E. coli contamination in fresh produce grown in semi-organic and organic farms.”

The article is:
Association of farm management practices with risk of Escherichia coli contamination in pre-harvest produce grown in Minnesota and Wisconsin


Use of animal wastes in fertilization of fresh fruits and vegetable plots significantly increased the risk of E. coli contamination in fresh produce grown in semi-organic (OR = 12.9, 95% CI = 2.9–56.3, P-value less than 0.0001) and organic (OR = 13.2, 95% CI = 2.2–61.2, P-value less than 0.0001) farms (Table 4). Although composting did not affect the E. coli prevalence in semi-organic and organic produce, ageing of non-composted manure for more than 6 months contributed to a significant reduction of the risk (OR = 4.2 95% CI = 1.7–12.3, P-value = 0.005) among organically produced fruits and vegetables. Semi-organic and organic farms used various types of animal wastes such as chicken, hog, horse and cattle manure. Among these different manure types, use of cattle manure for fertilization of produce crops increased the risk of E. coli contamination by approximately 7-fold (OR = 7.4, 95% CI = 1.6–36.8, P-value = 0.003). Farmers who used animal wastes for fertilization applied manure either in the Spring or in the Fall or both in Spring and Fall. However, these different times of application of manure application did not have any significant effect on the E. coli prevalence in semi-organic and organic produce.

4. Discussion
Several reports have found that some of the farm-level practices for handling and treating animal manure before their application as fertilizer might reduce the survival of contaminating bacteria in manure and in agricultural soil, in which manure had been amended as a fertilizer ([Hutchison et al., 2004], [Kudva et al., 1998] and [Lung et al., 2001]). Thus a major portion of the survey’s questionnaire was meant to collect information on manure-handling and treatment practiced by the semi-organic, organic and conventional produce growers who took part in this study.
During the two-year study, less than half of the conventional growers used animal manure for fertilization of their crops, while 70 to 90% of organic and semi-organic farmers applied animal manure for fertilization. This finding was expected as organic growers are not allowed to use most of the chemical fertilizers that conventional growers can use (AMS/USDA, 2000). Although the majority of the conventional farmers did not use animal waste as fertilizer, only 57% of the manure users composted animal manure before using it as fertilizer. As many as 70% of the semi-organic and almost all the organic farmers who used manure fertilizer applied composted manure.
Our findings, both during the present study and in a previous study, showed that only 1.5 to 2.5% of pre-harvest conventional produce had detectable E. coli contamination ([Mukherjee et al., 2004] and [Mukherjee et al., 2006]). This low number of positive samples prevented any risk-factor analyses involving conventional farms. However, among the 6 conventional farms which had at least one E. coli contaminated sample, 4 used manure-based fertilizers. In this study, only semi-organic and organic farms were included in the analyses of farm-level risk-factors associated with greater prevalence of E. coli. Among these two farm types, users of animal waste as fertilizer were at a significantly greater risk of E. coli contamination in their produce compared to those who did not use manure fertilizer (Table 4).
Ageing of animal wastes before their application as fertilizer was used by several of the participating farmers. Ageing of animal manure for less than 6 months increased the risk of E. coli contamination by more than four folds in organic produce (Table 4). However, this risk-factor did not show a significant effect on E. coli prevalence in semi-organic produce. Hutchison et al. (2004) reported that spreading livestock manure on the top of agricultural soil and let it age without mixing them into the soil significantly reduced counts of pathogenic bacteria such as E. coli O157:H7, Salmonella enterica, Listeria monocytogenes, and Campylobacter jejuni.


Association of farm management practices with risk of Escherichia coli contamination in pre-harvest produce grown in Minnesota and Wisconsin

Avik Mukherjee, Dorinda Speh and Francisco Diez-Gonzalez.

Food Science and Nutrition Department, University of Minnesota, St. Paul, Minnesota 55108, USA
South West Research and Outreach Center, University of Minnesota, Lamberton, Minnesota 56152, USA

International Journal of Food Microbiology
Volume 120, Issue 3, 15 December 2007, Pages 296-302

Microbiological analyses of fruits and vegetables produced by farms in Minnesota and Wisconsin were conducted to determine the prevalence of Escherichia coli in pre-harvest fruits and vegetables. During the 2003 and 2004 harvest seasons, 14 organic (certified by accredited organic agencies), 30 semi-organic (used organic practices but not certified) and 19 conventional farms were sampled to analyze 2029 pre-harvest produce samples (473 organic, 911 semi-organic, 645 conventional). Before each harvest season, a farmer survey was conducted to collect relevant information on farm management practices that might affect the risk of E. coli contamination in fresh produce. The use of animal wastes for fertilization of produce plants increased the risk of E. coli contamination in organic (OR = 13.2, 95% CI = 2.2–61.2, P-value less than 0.0001) and semi-organic (OR = 12.9, 95% CI = 2.9–56.3, P-value less than 0.0001) produce significantly. Improper ageing of untreated animal manure significantly increased this risk in organic produce (OR = 4.2 95% CI = 1.7–12.3, P-value = 0.005) grown using such manure as a fertilizer. Organic growers who used cattle manure for fertilization of their crops showed significantly greater risk of contamination with the E. coli (OR = 7.4, 95% CI = 1.6–36.8, P-value = 0.003), compared to those who used other types of manure-based fertilizer. In Minnesota, organic and semi-organic produce collected from the southeastern (SE) part of the state were at a significantly greater risk of E. coli contamination (OR = 3.45, 95% CI = 1.8–35.2, P = 0.008), compared to those collected from farms located in the southern (S) regions of the state. In Wisconsin, organic and semi-organic produce collected from the southern (S) cluster of farms were at approximately 3-times greater risk of E. coli contamination (OR = 2.67, 95% CI = 1.3–9.4, P = 0.004), compared to those grown in the northern (N) cluster of farms.

Keywords: Organic foods; Produce; Farm management; Pre-harvest practices; Risk-factors; Prevalence; Escherichia coli

Further relevant papers

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 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.
Bill Marler’s recent comments

Bad Seeds – Again – This time E. coli O104:H4

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
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.

Internalization of human pathogens within growing salad vegetables.
Warriner K, Ibrahim F, Dickinson M, Wright C, Waites WM.
Biotechnol Genet Eng Rev. 2003;20:117-34.
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G

Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts.
Itoh Y, Sugita-Konishi Y, Kasuga F, Iwaki M, Hara-Kudo Y, Saito N, Noguchi Y,
Konuma H, Kumagai S.
Appl Environ Microbiol. 1998 Apr;64(4):1532-5.

Department of Biomedical Food Research, National Institute of Infectious
Diseases, Shinjuku-ku, Japan.

Using cultivation, immunofluorescence microscopy, and scanning electron
microscopy, we demonstrated the presence of viable enterohemorrhagic Escherichia 
coli O157:H7 not only on the outer surfaces but also in the inner tissues and
stomata of cotyledons of radish sprouts grown from seeds experimentally
contaminated with the bacterium. HgCl2 treatment of the outer surface of the
hypocotyl did not kill the contaminating bacteria, which emphasized the
importance of either using seeds free from E. coli O157:H7 in the production of
radish sprouts or heating the sprouts before they are eaten.

Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in
growing bean sprouts.
J Appl Microbiol. 2003;95(4):719-27.
Warriner K, Spaniolas S, Dickinson M, Wright C, Waites WM.

Division of Food Sciences, School of Biosciences, University of Nottingham,
Leicestershire, UK.

AIMS: Investigate the interaction of bioluminescent Escherichia coli and
Salmonella Montevideo with germinating mung bean sprouts.
METHODS AND RESULTS: E. coli or Salm. Montevideo introduced on mung beans became 
established both internally and externally on sprouts after the initial 24 h
germinating period. In both cases the inoculated bacterium formed the predominant
microflora on the sprouted beans throughout. From the bioluminescent profile of
inoculated sprouting beans, bacterial growth was found to be in close proximity
to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low
viability were observed within the grooves between epidermal cells on hypocotyls.
Treatment with 20,000 ppm sodium hypochlorite removed the majority of bacteria
from the surface of hypocotyls although nonviable single cells were occasionally 
observed. However, viable bacteria were recovered from the apoplastic fluid, and 
extracts of surface-sterilized sprouts indicating that the internal bacterial
populations had been protected. This was confirmed using in situ
beta-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme
substrate (by the action of internalized E. coli) was visualized within the plant
vascular system.
CONCLUSIONS: E. coli or Salmonella present on seeds become internalized within
the subsequent sprouts and cannot be removed by postharvest biocidal washing.
SIGNIFICANCE AND IMPACT OF THE STUDY: Mung bean production should be carefully
controlled to prevent contamination occurring in order to minimize the health
risk associated with raw bean sprouts.

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.
US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA. [email protected]
[Salmonella are similar bacteria to E. coli in basic biological properties]
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.

Survival of Escherichia coli O157:H7 in the rhizosphere of maize grown in waste-amended soil.

Williams AP, Avery LM, Killham K, Jones DL.
School of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd, UK. [email protected]
J Appl Microbiol. 2007 Feb;102(2):319-26.
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.
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.
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.
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

Exploring the benefits of organic nutrient sources for crop production and soil quality.  Rosen, C.J. ; Allan, D.L. (2007)Horttechnology, 17, 4, pp  422-430

Soil quality is generally improved with application of organic nutrient sources, but careful management is required to avoid environmental risks of nitrate (NO3) leaching and phosphorus accumulation.
Provided that nutrient supply is equal, yields with organic sources tend to be similar to those with inorganic sources. However, lack of available nitrogen (N) that is synchronous with plant demand often limits yields in organic cropping systems. ….
Phytonutrient content also may be affected by differences in pest control strategies among cropping systems regardless of nutrient source. There is a slight, but significantly, increased risk of produce contamination by Escherichia coli and other enteric bacteria contamination on produce when organic nutrient sources are used, but if proper guidelines are followed, contamination with the lethal serotype O157:H7 does not appear to be a major concern. Appropriate management of organic inputs is critical to achieving potential benefits for crop production and soil quality.

One potential risk for people consuming produce grown with manure or manure-based composts is an increased exposure to enteric pathogens (Buck et al., 2003). The bacteria of concern are Escherichia coli, Salmonella spp., and Listeria monocytogenes. E. coli and Salmonella are in animal gastrointestinal tracts and consequently in manure, whereas L. monocytogenes is in decaying plant residues, soil, and animal manure. Consumption of produce contaminated with the E. coli serotype O157:H7 can in some cases cause death in people with weak immune systems. Numerous outbreaks of gastrointestinal disease have been linked to consumption of fresh fruit and vegetables; however, the source of contamination can occur at various stages of production through actual sale of the final product. Although organic nutrient sources are not the only source of bacterial contamination, precautions in handling manure and manure compost need to be taken. To reduce the risk of contaminated produce, certified organic methods require that manure applied to fields must either be properly composted before application or produce for human consumption cannot be harvested for at least 90 d after application for edible portions of the crop not in direct contact with the soil and 120 d for edible portions in direct contact with the soil (USDA, 2000). Specific procedures that maintain temperatures in the pile to kill the pathogens must be followed during the composting process…
 Loncarevic et al. (2005) reported that 8.9% of 179 organically grown lettuce samples collected from 12 producers in Norway were contaminated with E. coli, but only 2.2% of the samples were considered to have high enough levels to cause foodborne illness. The serotype O157:H7 and Salmonella were not detected in any of the samples. Listeria monocytogenes serogroups 1 and 4 were isolated from 1.1% of the samples. In a study to determine the potential for contamination with E. coli serotype O157:H7, Johannessen et al. (2005) transplanted lettuce seedlings into soil fertilized with manure inoculated with E. coli O157:H7. After 50 d, the serotype was not detected in any parts of the lettuce leaves or roots; however, indigenous E. coli was detected sporadically on lettuce leaves… Based on these studies, it appears that there is a slight, but significant, increased risk of produce being contaminated with E. coli and other enteric bacteria when organic nutrient sources are used. However, contamination with the O157:H7 serotype resulting from use of organic nutrient sources does not appear to be a major concern.

Lett Appl Microbiol. 2005;41(2):186-9.

Bacteriological quality of organically grown leaf lettuce in Norway.


Section for Feed and Food Microbiology, National Veterinary Institute, Oslo, Norway. [email protected]



To investigate bacteriological quality in organically grown leaf lettuce, including the presence of selected pathogenic bacteria, and to obtain information about organic lettuce production, including fertilizing regimes.


Altogether 179 samples of Norwegian organically grown lettuce were collected from 12 producers. Escherichia coli was isolated from 16 of the lettuce samples, but in 12 of these contamination was sufficiently low (less than 100 CFU g(-1)) that they would be considered to be of acceptable bacteriological quality. Escherichia coli O157 and Salmonella were not detected in any of the samples. Listeria monocytogenes serogroups 1 and 4 were isolated from two samples.


Organic lettuce produced in Norway was generally of acceptable bacteriological quality, but the results show that contamination of organic lettuce with E. coli and L. monocytogenes do occasionally occur.


These results suggest that organically grown lettuce may be contaminated with E. coli and L. monocytogenes during cultivation.





[PubMed – indexed for MEDLINE]
Food Microbiol. 2010 Aug;27(5):679-84. Epub 2010 Mar 25.

Microbiological quality of fresh lettuce from organic and conventional production.


University of Lleida, UDL-IRTA Centre, XaRTA-Postharvest, Rovira Roure 191, 25198-Lleida, Spain.


Previously there was no available information on the levels of indicator bacteria and the prevalence of pathogens in fresh lettuce grown in organic and conventional farms in Spain. A total of 72 lettuce samples (18 farms for 4 repetitions each) for each type of the agriculture were examined in order to assess the bacteriological quality of the lettuces, in particular the prevalence of selected pathogens. The lettuce samples were analyzed for the presence of aerobic mesophilic, psychrotrophic microorganisms, yeasts and moulds, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas spp. and presumptive Escherichia coli, Salmonella spp. and Listeria monocytogenes. The mean aerobic mesophilic counts (AM) were 6.35 +/- 0.69 log(10) cfu g(-1) and 5.67 +/- 0.80 log(10) cfu g(-1) from organic and conventional lettuce, respectively. The mean counts of psychrotrophic microorganisms were 5.82 +/- 1.01 log(10) cfu g(-1) and 5.41 +/- 0.92 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Yeasts and moulds (YM) mean counts were 4.74 +/- 0.83 log(10) cfu g(-1) and 4.21 +/- 0.96 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Lactic acid bacteria (LAB) were present in low numbers and the mean counts were 2.41 +/- 1.10 log(10) cfu g(-1) and 1.99 +/- 0.91 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Pseudomonas spp. mean counts were 5.49 +/- 1.37 log(10) cfu g(-1) and 4.98 +/- 1.26 log(10) cfu g(-1) in organic and conventional lettuce, respectively. The mean counts for Enterobacteriaceae were 5.16 +/- 1.01 log(10) cfu g(-1) and 3.80 +/- 1.53 log(10) cfu g(-1) in organic and conventional lettuce, respectively. E. coli was detected in 22.2% (16 samples) of organic lettuce and in 12.5% (9 samples) of conventional lettuce. None of the lettuce samples was positive for E. coli O157:H7, L. monocytogenes and Salmonella spp. From the samples analyzed by principal component analysis (PCA) a pattern with two different groups (conventional and organic) can be observed, being the highest difference between both kinds of samples the Enterobacteriaceae count.
Copyright 2010 Elsevier Ltd. All rights reserved.





[PubMed – indexed for MEDLINE]

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