SAMPLING AND ANAYSIS FOR
MICROBIAL CONTAMINAINTION OF
This module will cover…
• Outbreak case studies of four major
types dairy product pathogens
• Sampling for each of those pathogens
• Sample analysis using approved
• Resolution of the outbreak
This case history and lab methods
relate to Listeria monocytogenes
• Jan. – Aug. 1985: LA hospital notes an increased occurrence of
Listeriosis in pregnant women, apparently community-acquired.
• Initially 58 mother-infant pairs sickened. Mean maternal age 28
• One Caucasian and one Asian, all others (96%) were Hispanic.
Human Listeriosis in Hispanic Community
• Bacteria found in GI tracts
of humans, wild & domestic
animals, soil, water, foods.
• Can “hide” within white
• Can survive the low
• Disease Outbreaks are
typically associated with
contamination in the
Listeria monocytogenes residing within
white blood cells.
Major Listeria Outbreaks
• Disease Outbreaks typically associated with:
• processed ready-to-eat meats (hotdogs, deli-meats),
• sprouts & cantaloupe
• with soft cheeses (with unpasteurized milk).
Listeriosis: the Disease
• No or flu-like Symptoms
• Pregnancy-Associated Cases (≈ 20%)
• Invasive infections leading to
• abortion, stillbirth, newborn
• Non-Pregnancy Cases (≈ 80%)
• 3/4 are Immune-compromised
• Sepsis, meningitis, encephalitis
• Overall hospitalization rate ≈ 80%
• Leading cause of mortality among
food borne bacteria pathogens (20%)
• Compare fatality rates for E. coli,
Campylobacter and Salmonella of
about < 1 %
• Hispanic women 11 fold higher risk
Public Health Actions
• “Case Finding” with review of clinical and isolate records
from surrounding areas & states.
• Case-Control Studies using patient interviews and
• Product testing would be performed based on the results.
“Case Finding” Results
• 142 cases of Listeriosis
• 2/3 pregnancy related
• 1/3 non-pregnant adults
• 48 deaths
• 20 fetuses
• 10 neonates
• 18 non-pregnant adults
• (98% with preexisting
Premature infant delivery & death following
exposure to listeria in cheese. (1997 STOP)
Stillborn human infant with generalized
Listeria infection. (Vazquez, 2001)
Case-Control Study Results
• 1st Study: Patients 5.5 times more likely to have
consumed Mexican-style soft cheese.
• 2nd Study: Patients 8.5 times more likely to have
consumed Jalisco Brand Mexican-style soft cheese.
Queso fresco, a creamy,
soft, un-aged white cheese
Official BAM Culture Method
for Listeria monocytogenes
Following this we will return
with further results of the
outbreak investigation and
resolution of the case.
Raw Milk Testing for Listeria
5 to 7 days for negative
7 to 12 days for positive
Sample received from inspectors
Chain of Custody Form Completed
Submission Form Signed and Dated
Listeria enrichment broth –
FDA formula (LEB-FDA)
Cycloheximide, Acriflavin HCl, Nalidixic acid,
Sterile disposable beaker
Aseptically add approximately
25mL of liquid product to 225mL of
LEB-FDA. Mix well. Incubate LEB-
FDA with product aerobically at 29-
31°C for 4 hours
After 4 hours of pre-enrichment,
add LEB-FDA supplements and re-
incubate another 42-46 hours at
At 22-26 hours and 46-
50 hours, use a sterile
cotton-tipped swab to
inoculate LEB-FDA to
Oxford plates. Streak
the plates for isolated
colonies. Incubate the
Oxford plate aerobically
at 35-37°C for 46-50
Listeria colonies are
typically black with
a black halo (esculin
Gram positive rod
L. monocytogenes - Strong
hemolysis towards S. aureus
“umbrella motility” at 25°C
Biochemical Reactions for Listeria monocytogenes
Product Testing Results
• Cheese samples were collected from LA food markets.
• L. monocytogenes identified in unopened packages of
Jalisco brand cheese.
• Samples had different expiration dates suggesting a
on-going contamination problem.
Esculin positive Listeria colonies
• Day after Listeria culture from unopened product,
implicated cheese was recalled.
• Plant and Environmental Investigation:
• Multidisciplinary team bacteriologists, food
scientists, regulators, attorneys.
Further Public Health Actions
• Pasteurizer operating properly, but bypass possible.
• Records: on occasion more milk was received then
could be processed by pasteurizer.
• Alkaline Phos levels in cheese indicated insufficient
pasteurization on about 25% of production days.
• Environmental samples (vat and drain swabs) were
positive for Listeria epidemic strain phage-type.
• No Listeria organisms were found in raw-milk samples
from herds supplying the cheese factory.
Plant and Environmental Investigation
• However, the epidemic strain was
recovered in product from a separate
plant that shared the same source of
raw milk as the cheese plant.
Hypothesis for this contamination…
Milk by-passes pasteurization.
Pasteurization & Cheese
This concludes the presentation on
the investigation, sampling and
analysis of Listeria dairy outbreaks.