Pathogen Behavior in Cheese · • Safety of raw milk cheese is primarily determined by the...

Pathogen Behavior in

Cheese

Dennis D’Amico, Ph.D.University of Connecticut

Current pathogens of concern in cheese• Mycobacterium bovis/tuberculosis • Brucella species • Clostridium botulinum• Bacillus cereus• Yersinia enterocolitica• Campylobacter jejuni• Enterotoxigenic Staphylococcus aureus • Salmonella species • Pathogenic Escherichia coli • Listeria monocytogenes

Listeria monocytogenes• Occurs widely on dairy farm (ubiquitous)

• soil, vegetation, silage, feces and water

• Numerous animal species are susceptible to infection

• healthy asymptomatic animals can shed in feces

• Listeria mastitis is possible but rare• Average milk prevalence estimate: 2-4%• Persistent processing environment contaminant

• Grows at refrigeration temperatures

Listeria monocytogenes• Symptoms of listeriosis

• Non-invasive: none, fever, muscle aches, gastroenteritis • Invasive infection:

• Fever, muscle aches, headache, stiff neck, loss of balance, etc.• Immunocompromised: septicemia and meningitis• Pregnancy: miscarriage, stillbirth, premature delivery, or life-threatening

infection of newborn

• Infectious dose:• Immunocompetent: can be as high as >10 million cells• Immunocompromised: may be <1000 cells

• Mortality rate: 20-30%; • meningitis ~70%; septicemia ~50%, perinatal/neonatal infections >80%

Escherichia coli• Inhabit intestinal tract of humans and animals

• Many are harmless• Also various pathogenic types

• Shiga-toxin producing E. coli (STEC)• E. coli that acquired genes that encode Shiga-toxins• Enterohemhorrhagic E. coli (EHEC):

• subset of pathogenic STEC with increased virulence• EHEC includes several serogroups, notably O157:H7

• Several others are emerging including the “Big 6” • O26, O45, O113, O111, O121, O145

E. coli O157:H7

• Ruminants are major reservoir• May survive for extended periods outside host• Average milk prevalence estimate: 2-5%• Low infectious dose: <1000 organisms

• Some suggest as few as 1-10 cells

• Symptoms: onset 1-9 days (often 3-4) • Immunocompetent:

• severe cramping and diarrhea which is initially watery but can become grossly bloody

• occasional vomiting and/or fever • Immunocompromised:

• hemolytic uremic syndrome (HUS): renal failure and hemolytic anemia • Terminal in 3-5% of cases

Salmonella enterica• > 2,500 different types (serotypes)

• All are public health concern

• Inhabits the GI tract of warm-blooded mammals• Poultry, swine, livestock, birds, rodents, etc.

• Animals can acquire infection• shed large numbers in feces, milk, and saliva

• Average milk prevalence estimate: 2-5%• Symptoms: onset 6-72h, lasts 4-7 days

• Immunocompetent: diarrhea, fever, and abdominal cramps• Immunocompromised: may spread to blood stream and other body sites • Mortality: Generally less than 1%

• Infectious dose:• Immunocompetent: >100 - 10,000 cells• Immunocompromised: may be as few as 1 cell

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Pasteurized/thermized cheese

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Staphylococcus aureus

• Carried by 30-50% of humans (nose, hair, skin, etc.)• Common cause of contagious mastitis • Estimated prevalence:

• ~20-30% raw cow milk• ~30-40% of raw goat and sheep milk

• Produces enough heat stable toxin to cause illness when population exceeds ~100,000 CFU/ml or g

• Toxin survives pasteurization even if cells don’t

• Growth and toxin limited by active starter culture• Symptoms: Quick onset (1-7 hours), short duration (hours-3 days)

• Typical: nausea, vomiting, cramping, and diarrhea

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Toxin in pasteurized milk cheese

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Toxin in raw milk cheese

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Pathogen behavior in cheese

• Pathogen growth and/or survival is a function of several factors including:

• Pathogen and contamination level• Physiological state of the bacteria• Acidity and rate of acid development (starter activity) • Curd cooking time and temperature• Post-manufacturing conditions and recontamination• Biochemical changes during aging• Aging and storage temperature of the cheese• Composition of the cheese (pH, salt in moisture, water activity)

Hurdle processing

• Limiting growth and survival through a combination of intrinsic, extrinsic and processing factors

• Combination inhibit growth more than any factor alone

MicrobialSafety

MicrobialContamination

Temperature

Acidity

Salt

Moisture

Risk continuum: An example

• Category 1: combination of factors eliminate pathogens that may be present in raw milk

• Category 2: pathogens if present in raw milk may survive, product does not support growth

• Category 3: limited factors, if any, that inhibit survival and growth of pathogens

New Zealand Food Safety Authority, 2010;US FDA Listeria Risk Assessment ,2003

Milk

• Raw vs. pasteurized • Overall, raw milk does not appear to offer greater

protection • Raw milk may better inhibit pathogen growth during

acidification• Pathogens die-off slightly faster in pasteurized milk cheese• Slight differences “cancel each other out”

Kocharunchitt and Ross. 2014. Quantitative Assessment of Microbiological Safety of Raw milk Cheese Manufacturing.

Kocharunchitt and Ross. 2014. Quantitative Assessment of Microbiological Safety of Raw milk Cheese Manufacturing.

Pathogen

• E. coli:• may grow faster during acidification and curd formation• Rapid initial inactivation followed by slower phase

• Listeria:• Slow initial inactivation followed by more rapid phase

Kocharunchitt and Ross. 2014. Quantitative Assessment of Microbiological Safety of Raw milk Cheese Manufacturing.

Temp vs. growth rate• Optimum growth for most pathogens: ~37C (98.6F)

Temperature

• Pathogen growth ranges• Staph aureus: ~7-50 ∘C; toxin prod. 10-48 ∘C (45-122 ∘ F)• L. monocytogenes: −0.4-45 ∘C (<32-113 ∘ F)• Salmonella: ~5-47 ∘C (45-118 ∘ F)• Pathogenic E. coli: ~2-50 ∘C (36-122 ∘ F)

• Most curd cooking temps allow pathogen growth • Sublethal injury can occur at temps beyond

maximum growth temp.• Most pathogens start to die at temps >55-60C (131-140F)

Parmigiano Reggiano• Cooking and pressing of curd at high temps • 53-56C (127-133F) inactivates pathogens

Panari et al. 2001

Emmental• Cooking and pressing of curd at high temps • 52-54C (126-129F) inactivates various pathogens

Bachmann and Spahr, 1995

Tilsit• Cooked at 42C (107F) for 15 min

Pathogen variability:

Conditions that might kill one may not affect another

Aging and storage temp

• Pathogen inactivation rates are very slow • weeks to months to reduce by one order of magnitude

• If pathogen levels are decreasing during aging:• An increase of 5°C in maturation temperature

approximately doubles the inactivation rate

• If pathogen levels are increasing:• cheeses should be kept cold to limit growth • Finished RTE cheese must be refrigerated

Kocharunchitt and Ross. 2014. Quantitative Assessment of Microbiological Safety of Raw milk Cheese Manufacturing.

Aging and storage temp

Kocharunchitt and Ross. 2014.

Aging and storage temp

FIG. 1. (A) GROWTH OF LISTERIA MONOCYTOGENES INOCULATED IN QUESO FRESCO CURDS, FORMED INTO A CHEESE BLOCK, SLICED AND STORED AT 4 OR 10C. (Legget et al., 2012. Journal of Food Safety32: 236–245)

pH and acidification

• Optimum growth for most pathogens is ~pH 7• Pathogen growth ranges

• Staph aureus: ~4 - 10; toxin prod. limited <5• L. monocytogenes: 4.4 - 9.4• Salmonella: 3.7 - 9.5 • Pathogenic E. coli: 4 - 9

• Faster acidification impedes growth• If pH rises during aging and storage:

• Sublethally injured cells can repair and multiply • Healthy pathogens can multiply at faster rates

pH and acidification

• pH ranges of cheese

Kocharunchitt and Ross. 2014.

pH and acidification

• Ranges within product

Kocharunchitt and Ross. 2014. Quantitative Assessment of Microbiological Safety of Raw milk Cheese Manufacturing.

pH and acidificationEffect of final pH on Salmonella in Cheddar during aging

Ripened cheese• Increase in pH during ripening and aging• Promotes growth in core and on surface 1,2,3,4,5

• Aging inadvertently contributes to risk

1: Back et al., 1993;2: D Amico et al., 2008;

3: Gay and Amgar , 2005; 4: Genigeorgis et al., 1991; 5: Ryser and Marth, 1987.

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Ryser and Marth, 1987

Salt as NaCl• Salt is an important modulator of microbial activity• Maximum salt content tolerated by pathogens:

• Staph. aureus: 10-25%; toxin prod. limited >10%• L. monocytogenes: 10%• Salmonella: 8%• Pathogenic E. coli: 6.5%

• Most cheeses range from 1.6-2.5%• Salt is dissolved in the aqueous phase (SM)• SM for most cheeses range from 2.2-6.5%

• These levels can slow growth rate, sublethal injury

Water activity: aw

• Water in foods is often described as:• "free", "unbound", "available” OR• “bound”, “unavailable”

• Water activity (aw):• a measure of how efficiently the water present can take part in a

chemical or physical reaction

• Salt, sugar, and proteins can bind water making it no longer available to support microbial growth

Water activity: aw

• Minimum aw tolerated by pathogens:• Staph. aureus: 0.83; toxin prod. 0.85• L. monocytogenes: 0.92• Salmonella: 0.94• Pathogenic E. coli: 0.95

• Lower aw in cheese can slow growth rate

Water activity: aw

• Water activity ranges

Water activity: aw

Kocharunchitt and Ross. 2014.

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Pathogen growth on cheese

Bioprotection• Smear flora increases pH (~5 → 7)• E. coli O157:H7 population decreases over time

• coincides with ↑pH and presumably smear growth

Maher et al., 2001

General conclusions

• Safety of raw milk cheese is primarily determined by the hygienic quality of the milk, not by the ability of the process to eliminate pathogens

• Exceptions: some brined cheeses, cooked curd (e.g., hard grating cheese)

• Initial pathogen levels in milk must be below those known to be a threat to public health

• Initial cheesemaking steps increase pathogen levels by ~10-100+ times (~1-2.5 log CFU/g)

• Subsequent steps must reduce levels at least as much • Aging will help with some cheese, but not all