THE BOVINE MILK MICROBIOME

Mark McGuire

FLOW OF MILK FROM A FARM TO PROCESSOR

HOW TO ASSESS PRESENCE OF BACTERIA?Culture-dependent methods• Relies on specific culture media

conditions for specific bacterial genera/species (mostly pathogenic)

• Only see what you’re looking for

Culture-independent methods• Rely on molecular techniques and

specific primers

• Identification of bacterial taxa often related to genetic variation in 16S rRNAgene

Image:RodolfoParulan Photography

Tortoli (2003)ClinMicrobiol Rev16:319-354

“Hypervariable”

Conserved

Usingacombinationofbothtypesofmethodsisverypowerful.

COMMON BACTERIA GROWN FROM BOVINE MILK

PhotosfromJanetWilliams

Species Colonyforming unitsperml

LactococcusStreptococcusLactobacillusLeuconostocEnterococcus

8.2x101 to1.4x1041.4x101 to1.5x1041.0x102 to3.2x1049.8x101 to2.5x1032.6x101 to1.6x103

Quigleyetal(2013)FEMSMicroRev37:664-698

COMPARISON OF METHODS WITH MILK• Compared various papers in the

literature that used culture dependent (CD) or independent (next generation DNA sequencing; NGS) methods

• Not necessarily directly comparable• Assumes similarity across all

samples within a species

• Breadth of assessment limited in some cases

Quigleyetal(2013)FEMSMicroRev37:664-698

IS THERE A DIFFERENCE BEFORE AND AFTER MILKING?

Reynolds, Hunt, Williams and McGuire, unpublished

• 15cows• 2quarters• Usedaseptic

method• V1-V216SrRNA

SOURCES OF BACTERIA IN RAW MILK

Doyle et al (2016) Appl Environ Micro 83:e02694-16

• Irishdairy• Sampledbedding,feces,

grass,silage,soil,teatswabsandcomparedtoindividualcowandbulktankmilk

• V3-V416SrRNA

Bulktank

Cow

BeddingFecesGrassSilageSoilTeatUnknown

ARE THE BACTERIA FROM THE TEAT?

Reynolds, Hunt, Williams and McGuire, unpublished

Needleaspiratethroughsurgicallyscrubbedteatwall• 15cows• 2teats

No bacterial growth in 25-40% of milk samples from cows with clinical mastitis (Makovec and Ruegg (2003) J Dairy Sci 86:3466-3472; Bradley et al. (2007) Vet Rec 160:253-257; Taponen et al. (2009) J Dairy Sci92:2610-2617)§ Culture conditions are tailored to a pathogen of interest§ Conditions cannot meet the requirements of all microbes present§Nutrients; aerobic vs. anerobic growth§Biological functions of the host§<104 colony forming units/ml; culture 0.01 ml of milk; chance of growth?

Clearly there is a need for a method to identify bacteria without culture!

DOES BACTERIAL CULTURE ASSESS THE BIODIVERSITY PRESENT?

BOVINE MILK MICROBIAL COMMUNITY IN MILK WITHOUT GROWTH

Kuehn et al (2013) PLoS One 8:e61959

Culture-negativeclinical----------Healthy----------- Lowsomaticcell

▪ 3farms▪ 10cows▪ V1-V216SrRNA

Clinicalmastitiswithoutgrowthvs.healthywithinsamecow

Twoquarterswithlowsomaticcellcount(LSCC)

BOVINE MILK MICROBIAL COMMUNITY BY SOMATIC CELL COUNT

▪ 2farms▪ 177samples▪ V1-V216SrRNA

Healthy1=<20,000cells/ml2=21,000to50,000cells/ml3=>50,000cells/ml

SubclinicalCulturepositive4=>400,000cells/ml

5=Mastitisculturenegative

Oikonomou et al (2014) PLoS One 9:e85904

BOVINE MILK MICROBIAL COMMUNITY BY SOMATIC CELL COUNT

▪ 2farms▪ 103cowsbyquarter▪ V1-V316SrRNA

SomaticCellCount(SCC)Low=<200,000cells/mlMedium=200,000to400,000cells/mlHigh=>400,000cells/ml

Brooker and McGuire, unpublished

CLINICAL MASTITIS IN COWS

Reynolds, Hunt, Williams and McGuire, unpublished

BMM=bovinemilkmicrobiome

IS THERE A DIFFERENCE BY FARM?

Rodrigues et al (2017) J Dairy Sci 100:1-17

• Bulktankmilkfrom19farmsover2months

BACTERIA RELATED TO MILK QUALITY

Rodrigues et al (2017) J Dairy Sci 100:1-17

HigherSomatic CellCount HigherStandardPlateCountCorynebacteriumStreptococcusLactobacillusCoxiellaArthrobacterLactococcus

AcinetobacterEnterobacteriaceaeCorynebacteriumStreptococcus

VARIATION IN PREDOMINANT BACTERIA IN RAW MILK

Kable et al (2016) mBios 7:e00836-16

• TwodairyproductionfacilitiesinCalifornia

• 899tankertruckers• V416SrRNA

CORE RAW MILK MICROBIOME FROM TANKER TRUCKS

Kable et al (2016) mBios 7:e00836-16

• Foundinall899tankertrucks

• Also17othersbetween0.25and0.97%

FamilyorGenus % Relativeabundance

StreptococcusUnidentifiedClostridialesStaphylococcusUnidentifiedRuminococcaceaeCorynebacteriumTuricibacterUnidentified PeptostreptococcaceaeUnidentifiedLachnospiraceaeClostridiumUnidentifiedClostridiaceaeAcinetobacterUnidentifiedPlanococcaceae

6.516.335.454.353.702.452.222.031.471.331.191.09

PRESENCE OF PATHOGENSSouth Dakota and Minnesota

• 131 dairy herds• Bulk tank milk samples collected

using National Mastitis Council methods

Pathogen Percentofsamples

Campylobacter jejuniShigatoxin-producingEscherichiacoliListeriamonocytogenesSalmonellaYersiniaenterocolitica

9.23.84.66.16.1

26.7% of samples contained one or greater pathogens

Jayarao and Henning (2001) J Dairy Sci 84:2157-2162

PRESENCE OF PATHOGENSPennsylvania

• 248 dairy herds• Bulk tank milk samples collected

using National Mastitis Council methods

Pathogen Percentofsamples

Campylobacter jejuniShigatoxin-producingEscherichiacoliListeriamonocytogenesSalmonellaYersiniaenterocolitica

22.42.861.2

13% of samples contained one or greater pathogens

Jayarao et al (2006) J Dairy Sci 89:2451-2458

PRESENCE OF PATHOGENSNAHMS Dairy 2002

• 861 bulk tank milk samples• 21 states

Pathogen(cultured) Percentofsamples

ListeriamonocytogenesSalmonellaColiforms

2.02.495.0

Van Kessel et al (2004) J Dairy Sci 87:2822-2830Van Kessel et al (2005) J Dairy Sci 88:3475-3479

Pathogen(PCR) Percentofsamples

Salmonellaenterica 11.8

PRESENCE OF PATHOGENSNAHMS Dairy 2007

• 536 bulk tank milk samples• 519 in-line milk filters• Used PCR for S. enterica and

pathogenic E. coli• Used culture for L.

monocytogenes

Pathogen Percentofoperations

ListeriamonocytogenesSalmonellaentericaShigatoxin-producingEscherichiacoli

7.128.1

15.2(bulktank milk)51.0(filter)

Van Kessel et al (2011) J Food Prot 74:759-768

PASTEURIZED MILK ORDINANCE (PMO)• Total bacteria count leaving farm is <100,000 cfu/ml• Total bacterial count in commingled milk at the processor is <300,000

cfu/ml• Total somatic cell count is <750,000 cells/ml

Grade “A” Pasteurized Milk Ordinance 2015 Revision

PASTEURIZED MILK ORDINANCE• All raw milk shall be cooled to 10 ºC within 4 hours of 1st milking and to 7 ºC

or less, within 2 hours after completion• All farm bulk milk tanks have approved temperature-recording device

Grade “A” Pasteurized Milk Ordinance 2015 Revision

IS RAW MILK SAFE?“Usually, the bacteria in milk are harmless, and if this were always true there would be no reason to cool milk, except to delay souring. There is; however, no way for the dairy operator or regulating officer to be absolutely sure that no disease bacteria have entered the milk, even though observance of the other Items of this Ordinance will greatly reduce this likelihood. The likelihood of transmitting disease is much increased when the milk contains large numbers of disease bacteria. Therefore, it is extremely important for milk to be cooled quickly, so that small numbers of bacteria, which may have entered the milk, will not multiply.”

Grade “A” Pasteurized Milk Ordinance 2015 Revision, p. 59

IS RAW MILK SAFE?

Costard et al (2017) Emerging Infect Dis 23:957-964

• Risk associated with consumption of unpasteurized cow’s milk and cheese• Data from the National Outbreak Reporting System (US, 2009-2014)• Disease related to Shiga toxin-producing Escherichia coli, Salmonella spp.,

Listeria monocytogenes, and Campylobacter spp. from dairy products• 760 illnesses per year; 22 hospitalizations per year• Unpasteurized milk and cheese consumed by 3.2% and 1.6% of the

population, respectively, caused 96% of illnesses from contaminated dairy products

IS RAW MILK SAFE?

Costard et al (2017) Emerging Infect Dis 23:957-964

Unpasteurizeddairyproductscaused840timesmoreillnessand45timesmorehospitalizationsthanpasteurizeddairyproducts!

SUMMARY – MILK IS NOT STERILEWide variety of bacteria present in milk

§ At low concentrations <105 cfu/ml

Large variation in bacterial communities among cows, across farms, through processing• What are the major factors influencing the

variation?

Bacterial community changes with mastitis

Pathogenic bacteria are present in milk§ Factors driving presence have not been

clearly identified.

§ Also impacted throughout the milking system.

Pasteurizationisagreattooltominimizeriskoffoodborneillnessfrommilk!

THANK YOU!Michelle McGuireJanet WilliamsKatherine Hunt YahvahSarah BrookerSusan ReynoldsLarry Fox

SRA 2017ADVANCING THE SCIENCE WEBINAR SERIES CONTINUES:

1. Rodney Dietert (Cornell University), Protecting the Human Superorganism

(January 24)

2. Michelle McGuire (Washington State University), Human Milk: Mother Nature’s Prototypical Probiotic Food (March 21)

3. Mark McGuire (University of Idaho), Bovine Milk Microbiota: Insights and Perspectives from –Omics Studies (May 23, 5:30 EDT)

4. Anne Mendelson (Culinary Historians of New York), History of the Continuing Milk Wars (July 18, 5:30 EDT)

A panel of microbial risk assessors will deliberate evidence of microbiota influences on risk and benefit for fresh unprocessed and pasteurized milk (October, TBD)

prior to

SRA workshop and Round Table Panel Symposium (December 10-14, Arlington, VA).

MicrobiotaInformingNext-GenerationRisks&Benefits

SRA 2017Advancing the Science Webinar Series Continues:

1. Rodney Dietert (Cornell University), Protecting the Human Superorganism (January 24)

2. Michelle McGuire (Washington State University), Human Milk: Mother Nature’s Prototypical Probiotic Food (March 21)

3. Mark McGuire (University of Idaho), Bovine Milk Microbiota: Insights and Perspectives from –Omics Studies (May 23, 5:30 EDT)

4. Anne Mendelson (Culinary Historians of New York), History of the Continuing Milk Wars (July 18, 5:30 EDT)

A panel of microbial risk assessors will deliberate evidence of microbiota influences on risk and benefit for fresh unprocessed and pasteurized milk (October, TBD) prior to

SRA workshop and Round Table Panel Symposium (December 10-14, Arlington, VA).

Microbiota Informing Next-Generation Risks & Benefits