Shiga Toxin-Producing E. coli and Ruminant Diets: A Match

Made in Heaven?

Todd CallawayAgricultural Research Service/USDAFood and Feed Safety Research Unit

College Station, TX

Escherichia coli O157:H7 & friends• N. America has a VERY safe food supply, but it is still a focus of

concern• Enterhemorrhagic E. coli (EHEC)

– Bloody diarrhea, HUS, children– O157:H7, O26, O45, O103, O111, O121 and O145 are adulterants (FSIS) in

beef

• O157 evolved with cattle, lives in intestinal tract, adhering to intestinal mucosa– Recto anal junction, lymphoid tissue– 50,000+ years of evolution

• Depending on season 40 % of cattle may have O157 (non-O157 under investigation)– May be higher during summer, near 80%– “Supershedders” >105 CFU/g

Source: ASM

phuman infection = panimal infection x pcarcass contamination

x pproduct abuse x peaten by susceptible

= 0.05 x 0.4 x 0.02 x 0.2 = 0.00008 or24,800 cases/year in a population of 310 million

Thus reductions in pathogens before harvest can yield large returns in human health and reduced recalls

Focus on food safety “From Farm to Fork”

Processors say “below 103 cells/g, and we can take care of it”

Preharvest prevalence directly affects human illnesses

CarcassContamination

CarcassContamination

HideContamination

HideContamination

FecalShedding/

Super-Shedder

FecalShedding/

Super-Shedder

Environment

Fecal Microbiome

Host

Diet

Why should we worry about EHEC in live cattle?

• Several points for improvement– Food Safety (Reduced pathogen burden)

• Cross contamination of carcasses and foods

• Increased in-plant effectiveness

– Water Issues (indirect)• irrigation and run-off

– Spinach outbreak; Walkerton, ON

– Agricultural Fair/Open Farms (Petting zoo)/Farm worker safety (Keen et al., 2003)

• Dust from cattle and O157:H7 outbreak

Bovine Gastrointestinal Tract, ‘Generic’ E. coli, and E. coli O157:H7

Total Bacteria consortium (per gram digesta) > 2000 species1010 106 1010 1011

Total ‘generic’ E. coli (per gram digesta) 104 (104) (107-8) 107

E. coli O157:H7, when present (per gram digesta)<102 <102 <102 <106

Rumen, reticulum, omasum, and abomasum (fermentation reservoir)

Small Intestine Large Intestine

ColonCecum

Bovine Colonization and E. coli O157:H7

Colonization appears to be primarily at RAJ (rectoanal junction)

Infection in cattle is asymptomatic (lack of toxin receptors)

Seasonality and variable

Super-shedders (>104 CFU E. coli O157:H7 per gram feces) are a minor proportion

Need >20 animals to sustain levels of E. coli O157:H7 in the pen (recirculation)

Wells et al., 2008

• Management/ecological niche factors– Diet (including specific feeds and additives)

EffectsDistillers Grains Orange peel and pulp can reduce (essential oils)Forage feeding can alter microbiome and EHEC; forage quality

– Microbial Environment/stressCross-talk between host and microbesNorepinephrine role for “microbial organ”Microbiome and nutrient transporters and EHEC

What factors affect EHEC in cattle GIT?

Dietary factors in cattle• Co-evolution of host (lack of toxin receptors)• Fasting increases shedding of EHEC (Buchko)

and Salmonella (Grau)– Lack of VFA which are toxic to E. coli/Salmonella

• Barley increased EHEC shedding (Buchko)• Steam flaked corn increased EHEC relative to dry

rolled corn (Fox)• Monensin generally has no effect on EHEC

(Edrington, McAllister, Van Baale)– Increased dosage of 44 mg/kg did reduce (Paddock)

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RumenColon

E. c

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Grain (%)

Effect of Diet on intestinal coliform and E. coli populations

(Adapted from Diez Gonzalez et al., 1998)

-4 -2 0 2 4 6 8 10 12

Time (d)

Abrupt shift from 90% grain to 100% alfalfa hay

E. coli(Log10 cells/g)

98

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Bovine Diet and E. coli O157:H7: Forage vs Grain Diets

Wells et al, unpublishedWells et al, unpublished

In a on-going study, animals fed forage-based growing rations over 2 study years had higher fecal prevalence for E. coli O157:H7 than animals on corn-based finishing rations.

Long term feeding of forage-type diets may increase E. coli O157:H7.May be related to survival in manure and soils (Durso; Berry)

Forage quality appears to play a role in some form

DDGS impacts• Distillers grains are increasingly included in

animal rations – Significantly alter the gut microbiome (Pinchak)

• Increase fecal shedding of E. coli O157:H7 in cattle (Wells, 2009; Jacob, 2008)

• It appears that there is an effect of DG compared to corn on O157 populations, but magnitude is fairly small overall ~1.5 log10, but affects incidence– Unknown mechanism– Batch variability/QC issues

Fecal SamplesHigh Moisture/Dry-rolled Corn

Based Diet(0 or 40% WDGS)Fecal prevalence and percent of fecal

samples with enumerable E. coli O157:H7 were significantly different for the pens.

Fecal counts for E. coli O157:H7 were not significantly different at any measured level.

Bovine Diet and E. coli O157:H7: WDGS in Finishing Diets

Finishing Phase (0 or 40% WDGS) Hide Samples

Hide prevalence and percent of hide samples with enumerable E. coli O157:H7 were different for the pens.

Hide counts for E. coli O157:H7 were significantly different from40 CFU/100cm2 to 480 CFU/100cm2 .

Bovine Diet and E. coli O157:H7: WDGS in Finishing Diets

Wells et al, 2008Wells et al, 2008

Finishing PhasePrior to Shift

(0, 40 or 70% WDGS) Fecal Samples

Bovine Diet and E. coli O157:H7: WDGS in Finishing Diets

Wells et al, 2010Wells et al, 2010After Shift

Finishing PhaseTime after ShiftFecal Samples

Reductions in WDGS in the diet did not result in reduction of E. coli O157:H7 in feces by day 28, but did by day 56.

Bovine Diet and E. coli O157:H7: Shift WDGS from Finishing Diets

Wells et al, 2010Wells et al, 2010

The Bovine Fecal Microbiome: Corn vs WDGS

Finishing feedlot diets. N = 4, 0 or 40% WDGS

Shifts in Prevotella andBacteroides.

WDGS diet resulted in moreSporacetigenium andAnaerovorax

E. coli O157:H7 shedding not detected

Durso et al, 2010Durso et al, 2010

The Bovine Fecal Microbiome: Diet and OTUs Association

A B

Heifer-10

Steer-10

Steer-09

Steer-09Heifer-10

Steer-10

Principle Coordinate AnalysisWeighted UniFrac

Principle Coordinate AnalysisUnweighted UniFrac

Diet drives the fecal microbial composition in cattle

Heifer-1030% Alfalfa Haylage70% Corn Silage

Steer-0966% Corn26% Corn Silage8% Supplement

Steer-1083% Corn12% Corn Silage5% Supplement

Kim et al, submitted JASKim et al, submitted JAS

Red: Top 10% Average Enumerable E. coli O157:H7Blue: Lowest 10% Average Enumerable E. coli O157:H7

Community structure may not be driving shedding

Principle Coordinate AnalysisWeighted UniFrac

Heifers30% Alfalfa Haylage70% Corn Silage

Steers66% Corn26% Corn Silage8% Supplement

Steers83% Corn12% Corn Silage5% Supplement

Microbiome OTU Composition and Shedding of E. coli O157:H7

Kim et al, in preparationKim et al, in preparation

Specific Microbiome Taxa Association with E. coli O157:H730% Alfalfa Haylage70% Corn Silage

66% Corn26% Corn Silage8% Supplement

83% Corn12% Corn Silage5% Supplement

No obvious single taxa group driving shedding

No community association for heifers.Weak separation for steers diets

Community structure not driving supershedding

Few OTUs associated supersheddingfor any of the three diets

Kim et al, in preparationKim et al, in preparation

The Bovine Fecal Microbiome and E. coli O157:H7

Propensity to shed E. coli O157:H7 appears to associate with groups of OTUs within diet for animals fed finishing diets that are corn-based.

In contrast, there appeared to be little association between shedding E. coli O157:H7 and any single OTU or groups of OTUs for animals fed the corn silage/haylage diet.

Shedding high levels of E. coli O157:H7 appears to have low association with any OTU or groups of OTUs for any diet.

Tasco, tannins, and essential oils• Produced from brown seaweed

• Reduced growth of

EHEC and Salmonella in

In vitro fermentations

• Improves meat quality and shelf life

• Polyphenolics (tannins) can reduce EHEC– Naturally found in plants (Min; Lacombe)

• Essential oils can be bactericidal (spices)– Limonene, terpenes, thymol, oregano (Varel)

Dried orange peel/pulp• By-product, competitively prices into least-

cost ration formulation– Used widely in dairy and feedlot rations in

Florida and Southern California

• Orange oils (e.g., limonene) improve animal health and productivity (esp. in swine)

• Source of pectin and fermentable fiber to ruminal microbial population– 9% CP, TDN 82%, DE 1.64, NEm 0.88, and NEg 0.59

Mcal/lb (CSU)

Sheep fed 20% orangepeel consumed less peel than did sheep fed 10%

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5 and 10% OP reduced E. coli O157:H7 in rumen, cecum and rectum

Final pH , TVFA, and A:P ratio were not affected

Doesn’t work with Campylobacter

Dowd et al., 2006

Stress can affect microbial populationsStress can affect microbial populations

Bailey et al., 2011

Cecal community structurechanges followingsocial disruption (SDR)Inverse relationship bet.cytokines (IL-6) andbacterial populations

Oral administration of Norepinephrine

Pullinger et al., 2010

Stress is a complicated issueIt’s like pornographyHard to replicate

Host/bacterial crosstalk (Lyte) “Feed Me” signals Microbial organ as a driver

of homeostasis and disease

Conclusions• E. coli O157:H7 (and other EHEC?) co evolved with

cattle, so combatting it is neither easy, nor straightforward

• Diet impacts O157:H7 populations– Forage quality, WDGS vs corn, dry rolled vs steam-flake

• Diet changes the microbiome population• Diet-driven microbiota changes are not linked to

O157:H7 shedding shifts– Generic E. coli yes…

• What is driving the microbiome/EHEC change?– Future avenues for exploring this linkage are evolving rapidly

9th International Symposium on Shiga Toxin (Verocytotoxin)

Producing Escherichia coli Infections

Boston, Massachusetts

Todd Callaway

Jeff LeJeune

31 May- 3 June, 2015