Prevalence and Molecular Epidemiology ofClostridium difficile in Food and Companion Animals,

Retail Meats, and Humans in Minnesota

Dr Jeff Bender, DVM, MS, DACVPMProfessor, College of Veterinary MedicineCenter for Animal Health and Food Safety

Dr Timothy Snider, DVM, MPHInstructor, College of Veterinary MedicineVeterinary Population Medicine - Swine

Outline

• Background– Clostridium difficile infection – humans

• Study Objectives & Methods• Results• Discussion

Clostridium difficile• Anaerobic spore-forming bacillus • Identified as the causative agent of

pseudomembranous colitis in 1978

Pseudo-membranous

colitis

Healthy colon

Clostridium difficile• Fecal-oral transmission through contaminated

environment and hands of healthcare personnel• Antimicrobial exposure is major risk factor for

disease - Acquisition and growth of C. difficile- Suppression of normal flora of the colon

• Fluoroquinolones, Clindamycin, Penicillins, and Cephalosporins

The Problem• C difficile infections (CDI) are one of the most

common organisms causing healthcare associated infections (HA-CDI)

• HA-CDI has an estimated additional patient cost of $3,000 to $15,000 per hospitalization

• CDI with onset outside of hospital setting is common (Community Acquired Or CA-CDI)

Increase in CDI Severity• There has been an increase in CDI severity –

associated with a specific strain (NAP1/027)• This new strain produces more toxin A and B

than other strains of CD• Also produces a third toxin (Binary toxin)

Risk Factors• Antibiotic therapy – (strongest link with

fluoroquinolones)• Age greater than 65 years• Underlying illness• Naso-gastro intubation• Anti-ulcer medications• Long hospital stay

Recent Development

• The past decade has seen an increase in the cases of CDI outside of the hospital setting

• “Community Associated CDI” OR “CA-CDI”• CA-CDI missing some of the normal risk

factors: no recent history of AB txmts or hospital stay.

Why this development?Hypothesis:

– Animal contact and the consumption of food derived from animals thought be a potential source of CD exposure

Question:– Could animals or food sources play a role in the

increase of CA-CDI?

Objectives

1) Determine the frequency of CD in a convenience sample of retail meats & food animals (with and without diarrhea) in Minnesota (focus on central MN)

Objectives

2) Determine genotypes – For human CA-CDI, animal, and meat source CD isolates

3) Compare & contrast genotypes– Focus on Swine comparison for this presentation

Methods – Sample Collection• 104 fecal samples from human CA-CDI

• MDH’s human surveillance project – Central MN focus

• 600 fecal samples from animals• Both with and without diarrhea• Effort to focus solely on Minnesota sourced samples• VDL cases, VMC teaching hospital, farm samples, research trial animals

• 300 Retail Meat Samples• Targeted “Locally grown”, “Organic Certified”, “Antibiotic free”• Raw samples and “Ready to Eat” cured samples• Focus on small retail from Central MN (butcher shops and local sources)

Methods – Characterization• Pulse Field Gel Electrophoresis (PFGE)

– Standardized CDC protocol for PFGE and NAP type assignment– Nomenclature: NAP1 = North American Pulsotype 1

• Toxinotyping– PCR analysis of pathogenicity locus

• Binary Toxin PCR– PCR analysis of binary toxin locus

• tcdC gene sequencing– evaluate deletion status (associated with virulence)

Results: Sample Breakdown and CD+ Status

Species Samples Submitted

Number of Sources

SamplesC. difficile Positive

SourcesC. difficile Positive

Porcine 169 56 25 (15%) 14 (25%)

Bovine 227 89 10 (4%) 10 (11%)

Equine 50 50 7 (14%) 7 (14%)

Feline 48 37 5 (10%) 5 (14%)

Canine 49 39 4 (8%) 4 (10%)

Source = a single collection from a premises

Results - Comments

AGE• Majority of samples from cattle and swine were

young animals (pre-weaned, weaned, or juvenile)• Majority of the companion animal (cats, dogs, and

horses) samples were from mature animals

Results - Comments

Clostridium difficile Isolation

• All samples 51 animals were CD+ (51/560 = 9%)

• MN only samples 41 animals were CD+ (41/547 = 7.4%)

Results - CommentsDiarrhea Statistics• 65 animals had recorded diarrhea (65/560 = 12%)

• Recorded % with diarrhea varied by animal species (range 0 - 14%)

• CD+ rate was higher among animals with diarrhea (22%) versus healthy animals (7%) (P<0.001)

Genotype CharacterizationMinnesota Samples Only (41/547 CD+)

Characterization Number Percent

Non Toxigenic 12 29%

Toxigenic 29 71%

Genotype CharacterizationMinnesota Samples Only (41 CD+)

Species # Sources

# CD+ # Toxigenic

Percent

Porcine 56 17 (30%) 14 82%

Bovine 89 9 (10%) 8 89%

Equine 50 6 (12%) 4 67%

Feline 37 5 (14%) 1 20%

Canine 39 4 (10%) 2 50%

Human vs Porcine – Toxigenic

SpeciesCharacterization

Type TotalNumber Positive

Percent Positive

HumanToxigenic 104

7 7%

PorcineToxigenic 17

14 82%

Genotype CharacterizationMinnesota Samples Only (41/547 CD+)

NAP Type Number Percent

7 22 54%

4 3 7%

11 3 7%

6 2 5%

Unnamed 11 27%

Human vs Porcine – NAP shared

SpeciesCharacterization

TypeTotal

Number Positive

Percent Positive

Human NAP 7 104 4 4%

Porcine NAP 7 17 13 76%

Human NAP Unnamed 104 40 38%

Porcine NAP Unnamed 17 4 24%

Human Only – NAP

SpeciesCharacterization

TypeTotal

Number Positive

Percent Positive

Human NAP 4 104 18 17%Human NAP 1 104 11 11%Human NAP 11 104 11 11%Human NAP 6 104 6 6%Human NAP 12 104 5 5%Human NAP 2 104 4 4%Human NAP 9 104 3 3%Human NAP 10 104 2 2%

Genotype CharacterizationMinnesota Samples Only (29 Toxigenic CD+)

Characterization Number Percent of Toxigenic

Binary Toxin Positive

18 62%

Toxinotype V 22 76%

Toxinotype O 6 24%

tcdC (39 bp deletion)

14 48%

Human vs Porcine – Binary Toxin

SpeciesCharacterization

TypeTotal

Number Positive

Percent Positive

Human Binary Toxin 104 24 23%

Porcine Binary Toxin 17 10 59%

Human vs Porcine – Toxinotyping

SpeciesCharacterization

Type TotalNumber Positive

Percent Positive

Human Toxinotype 0 104 62 60%

Porcine Toxinotype 0 17 1 6%

Porcine Toxinotype V 17 13 76%

Human vs Porcine – tcdC

SpeciesCharacterization

Type TotalNumber Positive

Percent Positive

Human tcdC 0bp 104 75 72%

Porcine tcdC 0bp 17 1 6%

Porcine tcdC 39bp 17 8 47%

Discussion• Unique collaborative approach• First major effort to look at human,

animal and food sources regionally in Minnesota

• No CD was detected in food samples

Discussion• CD was identified in animals• No highly pathogenic NAP1/27 was

identified in animals• A wide variety of Toxinotypes and

Pulsotypes were identified

DiscussionPorcine Related Observations• Relatively high percent toxigenic (82%)• One similar NAP type with human isolates (NAP7)• Binary Toxin present (59%)• Toxinotype V (76%) and not present in human samples• tcdC 39bp (49%) and not present in human samples

Limitations

• Caution with extrapolations re prevalence– Convenience sample targeting younger

animals– Target was to find CD not determine herd or

animal prevalence

Special Thanks to– Funding provided by the HFHL Institute (UMN)

InvestigatorsUMN School of Medicine• James Johnson (PI)• Megan Shaughnessy

Minnesota Dept of Health• Stacy Holzbauer• Kirk Smith• Dave Boxrud• Elizabeth Cebelinski

UMN Food Science & Nutrition• Francisco Diez-Gonzalez• Rocio Sepulveda

Center for Animal Health and Food Safety (CAHFS)• Jeff Bender • Tim Snider

AcknowledgementsVeterinary Diagnostic Lab• Ron Joki• Sarah Opitz• Marie Gramer• James Collins• Kurt Rossow• Edgar d'Almeida• Christina Valeri

Veterinary Medical Center• Leslie Hiber

Post Graduate Students• Lisa Wolff (Dairy Samples)• Carmen Alonso• Carlos Andres Diaz• Marie Jose Clavijo• Matt Allerson• Nubia Macedo• Victor Niera

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