Transcript Elissa Redmiles and Lucy Erickson

Transient flora on hands as a
vehicle of transmission to food
for
Staphylococcus aureus
Elissa Redmiles and Lucy Erickson
Background

History of food bourn illness

Hypothesis
History of food borne illness


Since the beginning of human history, food borne
illness has been an issue
Various food preservation methods have been
employed over the years to slow down food spoilage
by microbes and natural aging processes, some
common prevention methods include:
 drying
 freezing
 freeze-drying
 salting
 curing
 canning
 pickling
 irradiation
 treating with sugar
 treating with inert gases (such as carbon dioxide)
History of food borne illness (cont.)

Transient flora found on human skin and hands plays an
important role in food contamination
 When those involved in food preparation fail to observe
hygienic methods
Hypothesis


It is hypothesized that Staphylococcus aureus, a
microbe occurring naturally on the hands of some
people, would be found at various locations throughout
the Microbiology building
It was also hypothesized that many different types of
bacteria would be isolated from the various locations
tested
Hypothesis (cont.)

The isolation of bacteria from
the inside handle of the
bathroom door particularly
holds implications for food
hygiene, as ideally it should
only be touched by pristinely
washed hands.
Protocol Design and Detailed Methods



Overall approach and rationale for
approach
Detailed methods and rationale behind
methods
Expected results
Overall approach and rationale for
approach

There were 2 objectives for this experiment:


To determine the number of different types of bacteria that
live on each of 7 objects tested
To identify on which objects
Staphylococcus aureus is
present
Overall approach and rationale for
approach (cont.)

To achieve these objectives a growth medium was
required
 Both agar and broth were considered
 Agar was determined to be a more suitable growth
medium since:
 It allows direct observation of different types of
bacterial colonies
 It prohibits the number of bacteria on an object from
being determined
 Broth was determined to be a less suitable growth
medium because:
 It allows the number of bacteria on an object to be
determined
 It does not allow direct observation of different types
of bacterial colonies
Overall approach and rationale for
approach (cont.)

To achieve the first objective a sampling technique was
required
 Both saline wetted and dry sterile cotton swabs were
considered:
 Wet cotton swabs were determined to be most effective
for obtaining the largest sample of microorganisms
 Microorganisms adhere more readily to wet surfaces
 A swab and streak method was used to inoculate the agar
plates
Overall approach and rationale for
approach (cont.)

To achieve the second objective a method of
differentiating Staphylococcus aureus was needed:
 Differential mediums and assays were used:
 The gram stain was used to differentiate gram negative
organisms from gram positive organisms
 The catalase assay was used to differentiate
Staphylococci (positive result) from Streptococci (negative
result)
 Blood Agar was used to differentiate S. aureus (positive
result) from S. epidermis (negative result)
 Mannitol Salt Agar was used to differentiate S. aureus
(positive result) from S. epidermis (negative result)
 The CoAgulase assay was used to differentiate S. aureus
(positive result) from S. epidermis (negative result)
Detailed Methods



Day 1
 Made Mannitol Salt Agar
 Autoclaved and cooled medium
Day 2
 Poured Mannitol Salt Agar plates
Day 3
 Obtained Trypticase Soy Agar (TSA) plates
 Obtained a package of sterile cotton swabs, and a tube of
diluted saline
 Swabbed 7 objects:
 Bathroom sink faucet (1st floor)
 Bathroom (interior) doorknob (1st floor)
 Micro bench sink faucet (Room x)
 Front door handle—Micro building
 Handicapped door button—Micro building
 Computer mouse in lab
 Stair rail (1st floor)
Detailed Methods (cont.)

Day 3 (cont.)
 Swabbing method used:
 Dipped tip of sterile swab into saline
 Swabbed 1 of 7 objects
 Struck inoculated swab on sterile agar plate with zigzag
motion
 A new swab was used for each plate
 4 plates (2 TSA and 2 Mannitol Salt Agar) were used for
each object
 Discarded the swab
 Incubated all plates at 37°C for 48 hours
Detailed Methods (cont.)

Day 4
 Observed growth of different colony types on TSA plates
 Observed growth of colonies on Mannitol Salt Agar plates
 Observed fermentation
 Photographed certain colonized plates
 Counted and recorded different types of colonies
 Selected different types of colonies from each Mannitol Salt
Agar plate
 Struck the selected colonies on (new) Mannitol Salt Agar
plates, on (new) TSA plates, and on (new) Blood Agar
plates
 Incubated all plates at 37°C for 24 hours
Detailed Methods (cont.)


Day 5
 Observed Mannitol Salt Agar plates
 Observed fermentation
 Observed TSA plates
 Observed Blood Agar plates
 Observed hemolysis
 Performed catalase assay on pure
colonies isolated from Mannitol Salt Agar plates
 Performed gram stain on pure colonies isolated from
Mannitol Salt Agar plates
 Performed CoAgulase assay on certain pure colonies isolated
from Mannitol Salt Agar plates
 Bactistaph latex 150 test
 Photographed relevant results
Day 6
 Observed results from CoAgulase assay
Detailed Rationale Behind Methods

Day 3
 Saline was used:
 To maintain highest degree of sterility
 Since bacteria adhere more readily to wet surfaces than
to dry surfaces
 In a diluted form so growth of non-salt tolerant
organisms was uninhibited
 A new swab was used for each plate to reduce the risk of
contamination by organisms not found on objects of interest
 TSA plates were used:
 As a control
 To determine the different types of organisms on objects
of interest
Detailed Rationale (cont.)

Day 3 (cont.)
 Mannitol Salt Agar plates were used:
 To isolate Staphylococcus aureus from objects of interest
 Staphylococcus aureus is a salt tolerant organism that
grows on Mannitol Salt Agar while other non-salt
tolerant organisms will not grow
 All plates were incubated at 37°C for 48 hours:
 37°C is in the optimal growth temperature range for
organisms that could be found on objects of interest
 48 hours is in the optimal growth time range for
organisms that could be found on objects of interest
 4 plates (2 TSA and 2 Mannitol Salt Agar) were used:
 To safeguard from error
 If an error occurred with one “set” (1 TSA and 1
Mannitol Salt Agar plate) results could be obtained
from another “set” (theoretically void of error)
Detailed Rationale (cont.)

Day 4
 Different colonies from each TSA plate were struck on (new)
TSA plates to confirm that colonies selected were pure and
different from other colonies on the plate
 Struck colonies from Mannitol Salt Agar on (new) Mannitol
Salt Agar plates to isolate pure colonies for further tests and
to confirm that colonies isolated were Staphylococcus aureus
 Staphylococcus aureus ferments Mannitol producing a
yellow zone surrounding colony
Detailed Rationale (cont.)

Day 4 (cont.)
 Struck colonies from Mannitol Salt Agar on (new) TSA plates
as a control

Isolated colonies were struck on Blood Agar to confirm that
isolated colonies were Staphylococcus aureus
 Staphylococcus aureus has -hemolysis on Blood Agar
Detailed Rationale (cont.)

Day 5
 Catalase test was performed to:
 Confirm that cultures from Mannitol Salt Agar plates were
Staphylococci (positive) not Streptococci (negative)
 Indicating that cultures were Staphylococcus aureus
 Gram stain was performed to:
 Confirm that cultures from Mannitol Salt Agar plates were
gram positive not gram negative
 Indicating that cultures were Staphylococcus aureus
 Confirm that bacteria colonized on Mannitol Salt Agar
plates formed clusters not chains
 Indicating that cultures were Staphylococcus aureus
Detailed Rationale (cont.)

Day 5 (cont.)

Bacti Staph Latex 150 test was performed to:

Confirm that isolated colonies tested positive not negative
for agglutination

Indicating that the cultures were Staphylococcus
aureus

CoAgulase assay was performed to:

Confirm that isolated colonies were CoAgulase positive
not CoAgulase negative

Indicating that the cultures were Staphylococcus
aureus
Expected Results

It was expected that:


Numerous types of microorganisms would live on the
tested objects
Staphylococcus aureus is found on some human
hands and would therefore be present on some of the
tested objects
Results

Findings

Summary of Findings

Relationship of findings to expectations
Findings




TSA plate 1 was inoculated with microorganisms from
the bathroom sink faucet:
 7 small white colonies grew
 8 large white colonies grew
TSA plate 2 was inoculated with microorganisms from
the bathroom sink faucet:
 16 small white colonies grew
 31 large white colonies grew
Mannitol Salt Agar plate 1 was inoculated with
microorganisms from the bathroom sink faucet:
 2 small white colonies grew
Mannitol Salt Agar plate 2 was inoculated with
microorganisms from the bathroom sink faucet:
 No growth
Findings (cont.)




TSA plate 1 was inoculated with microorganisms from
the computer mouse:
 No growth
TSA plate 2 was inoculated with microorganisms from
the computer mouse:
 1 small white colonies grew
 1 medium white colonies grew
Mannitol Salt Agar plate 1 was inoculated with
microorganisms from the computer mouse:
 2 small white colonies grew
 3 large peaked colonies grew
Mannitol Salt Agar plate 2 was inoculated with
microorganisms from the computer mouse:
 No growth
Findings (cont.)




TSA plate 1 was inoculated with microorganisms from
the stair rail:
 No growth
TSA plate 2 was inoculated with microorganisms from
the stair rail:
 No growth
Mannitol Salt Agar plate 1 was inoculated with
microorganisms from the stair rail:
 1 small white colony grew
 1 large colony with yellow zone grew
Mannitol Salt Agar plate 2 was inoculated with
microorganisms from the stair rail:
 No growth
Findings (cont.)




TSA plate 1 was inoculated with microorganisms from
the handicapped button:
 1 small yellow colony grew
TSA plate 2 was inoculated with microorganisms from
the handicapped button:
 1 small white colony grew
Mannitol Salt Agar plate 1 was inoculated with
microorganisms from the handicapped button:
 No growth
Mannitol Salt Agar plate 2 was inoculated with
microorganisms from the handicapped button:
 No growth
Findings (cont.)




TSA plate 1 was inoculated with microorganisms from
front door knob:
 33 small yellow colonies grew
 1 medium white colony grew
TSA plate 2 was inoculated with microorganisms from
the front door knob:
 1 small white colony grew
Mannitol Salt Agar plate 1 was inoculated with
microorganisms from the front door knob:
 No growth
Mannitol Salt Agar plate 2 was inoculated with
microorganisms from the front door knob:
 No growth
Findings (cont.)




TSA plate 1 was inoculated with microorganisms from
bathroom door knob:
 3 small yellow colonies grew
 1 small white colony grew
 1 medium white colony grew
TSA plate 2 was inoculated with microorganisms from
the bathroom door knob:
 7 small white colonies grew
 9 large white colonies grew
Mannitol Salt Agar plate 1 was inoculated with
microorganisms from the bathroom door knob:
 2 medium colonies with yellow zones grew
 2 large peaked colonies grew
Mannitol Salt Agar plate 2 was inoculated with
microorganisms from the bathroom door knob:
 No growth
Findings (cont.)




TSA plate 1 was inoculated with microorganisms from
lab sink:
 No growth
TSA plate 2 was inoculated with microorganisms from
the bathroom door knob:
 No growth
Mannitol Salt Agar plate 1 was inoculated with
microorganisms from the bathroom door knob:
 1 small white colony grew
Mannitol Salt Agar plate 2 was inoculated with
microorganisms from the bathroom door knob:
 No growth
Findings (cont.)


A Mannitol Salt Agar plate was inoculated with
microorganisms from the bathroom door handle:
 Growth and fermentation on Mannitol plate
 Gram positive rods
 Snapping replication was observed
 Catalase positive
A Mannitol Salt Agar plate was inoculated with
microorganisms from the bathroom sink:
 Growth and fermentation on Mannitol plate
 Gram positive cocci organized in grape like clusters
 Catalase positive
Findings (cont.)


A Mannitol Salt Agar plate was inoculated with
microorganisms from the stair rail:
 Growth and fermentation on Mannitol plate
 Gram positive rods
 Snapping replication was observed
 Catalase negative
A Mannitol Salt Agar plate was inoculated with
microorganisms from the computer mouse:
 Growth and fermentation on Mannitol plate
 Gram positive rods
 Snapping replication was observed
 Catalase positive
Findings (cont.)




A Blood Agar plate was inoculated with microorganisms
from the bathroom door handle:
 Growth on Blood Agar
 β-hemolysis
A Blood Agar plate was inoculated with microorganisms
from the bathroom sink:
 Growth on Blood Agar
 β -hemolysis
A Blood Agar plate was inoculated with microorganisms
from the stair rail:
 Growth on Blood Agar
 β-hemolysis
A Blood Agar plate was inoculated with microorganisms
from the computer mouse:
 Growth on Blood Agar
 α-hemolysis
Summary of Findings

Microorganisms isolated from the objects exhibited a
variety of phenotypes when grown on TSA plates:



Small white bacterial colonies

Small yellow bacterial colonies

Medium white bacterial colonies

Large white bacterial colonies
The phenotypes above indicate that a very diverse bacterial
population is present on the tested objects
This data supports the first hypothesis that numerous
types of microorganisms would live on tested objects
Summary of Findings (cont.)

Microorganisms isolated from the:


Stair rail

Computer mouse

Bathroom door handle
Are suspected to be Cornyebacteria since:

They undergo snapping
replication


Form “Chinese letters”

Are gram positive rods
This was not hypothesized
Summary of Findings (cont.)

Microorganisms isolated from the bathroom sink are
suspected to be Staphylococcus aureus
 The samples are strongly suspected to be Staphylococcus
aureus since:
 They are gram positive cocci seen in grape-like clusters
 Are catalase and CoAgulase positive
 Have β-hemolysis
 Ferment Mannitol
 These phenotypes are identical
to those exhibited by
Staphylococcus aureus

This supports the second hypothesis: Staphylococcus
aureus is found on human hands and would therefore
be present on some of the tested objects
Discussion


Description of Staphylococcus aureus
Why Staphylococcus aureus was expected
to be found

Incidence of disease

Virulence of Staphylococcus aureus

Comments on protocol

Comments on results

Comments on overall significance of project
Description of Staphylococcus aureus

Phenotype

Gram positive cocci

Found in pairs, chains, or grapelike-clusters

Produces heat stable enterotoxin

Salt tolerant

Ferments Mannitol

Catalase positive

CoAgulase Positive
Description of Staphylococcus aureus
(cont.)

Habitat
 Air
 Dust
 Sewage
 Environmental surfaces
 50% of humans (higher in health professionals)
 Foods
 Poultry and egg products
 Egg, tuna, potato, and chicken salads
 Milk and dairy products
Why Staphylococcus aureus was expected
to be found

Expected to find Staphylococcus aureus various surfaces
throughout the microbiology building where the transient
flora from many peoples’ hands has been transferred. One
of the places swabbed was the inside bathroom door handle,
where hopefully only people with freshly washed hands are
touching. Unfortunately, this is not always the case, and a
diverse population of bacteria were isolated from the door
handle. Someone who fails to wash his or her hands upon
exiting the restroom is unlikely to remember to wash them
before eating or preparing food for another person. Thus, if
S. aureus were found on the locations tested it could hold
strong ramifications for general human cleanliness, and
subsequently, health.
Why finding Staphylococcus aureus was
probable


Staphylococcus aureus is a component of the normal
flora for 50% of all people
 Carriers of Staphylococcus aureus transfer the bacteria to
every touched object
 Regardless of hand washing
 In proper growth conditions the bacteria will propagate
on objects such as those tested
 As Staphylococcus aureus contaminated objects are used,
bacteria are transferred to a users transient flora
Staphylococcus aureus was isolated on tested objects
 Thus, hand washing does not preclude the transfer of
Staphylococcus aureus to food
Incidence of disease

The incidence of illness is difficult to pinpoint due to a
number of reasons

Poor response from victims during interviews

Misdiagnosis (symptoms are very similar to those of Bacillus
cereus toxin)

Inadequate collection of lab samples

Improper lab examination
Virulence of Staphylococcus aureus

Causes staphyloenterotoxicosis (food poisoning)

Operates by toxin, not colonization

ID50 – 1.0 microgram

Onset of illness is rapid and varies based on:

Amount of food ingested

Amount of toxin present in food

Individual susceptibility

General health of victim
Virulence of Staphylococcus aureus
(cont.)

Symptoms:

Nausea

Vomiting

Abdominal cramping

Headache

Changes in pressure and blood rates

Average recovery time for mild case:

2-3 days
Virulence of Staphylococcus aureus
(cont.)

Treatment

Rest

Fluids

Medicines to calm stomach

Hospitalization/Intravenous therapy for more severe cases
(usually elderly or infants)
Virulence of Staphylococcus aureus
(cont.)

Prevention

Wash hands and under nails vigorously and often

Do not prepare food with an eye or nose infection

Do not serve food to others with open wounds

Keep food preparation areas sanitary


Store cooked food in wide, shallow containers and
refrigerate
Do not leave food out for long periods of time

Keep hot foods hot (above 140 degrees F)

Keep cold foods cold (40 degrees F or lower)
Comments on Protocol



Method of multiple plate inoculation was successful
 By using 4 plates for each object tested it allowed for
confirmation of results
Saline wetted swab and streak inoculation method was
successful:
 Isolated colony growth occurred
Testing and differential media:
 Catalase
 CoAgulase
 Mannitol
 Blood

Comments on Results
Comments on Results

Found numerous colonies resembling Corynebacteria, a
Gram-Positive rod generally found in soil




Methylene blue staining did not reveal metachromatic granules,
however not all species exhibit this trait
This finding is consistent with concept of bacteria found on
unwashed hands
Unable to identify specific strain of Corynebacteria isolated, since
many of the organisms can not be typed easily. Although there
have been significant advances in PCR technology, this
technology was not available.
Isolate from bathroom sink faucet is indicated by tests to
be Staphylococcus aureus
Comments on overall significance of project

This project aims to raise public awareness that
pathogenic microorganisms are present on numerous
objects touched prior to and after hand washing
References

Boyce, J., Pittet, D. (2002). “Guideline for Hand
Hygiene in Health-Care Settings” 1 April 2006. Morbidity
and Mortality Weekly Report, 51-RR16. 1-44
<http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5116a1.
htm>

CDC. Outbreak of community-associated methicillinresistant Staphylococcus aureus skin infections---Los
Angeles County, California, 2002--2003. Morbidity and
Mortality Weekly Report 2006;52:88.
<http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5512a
1.htm>

U.S. Food and Drug Administration. “Staphylococcus
aureus.” 1992. Food borne Pathogenic Microorganisms
and Natural Toxins Handbook.
<http://www.cfsan.fda.gov/~mow/chap3.html>
References



Rollins, David M. "Corynebacterium Summary." BSCI 424
— Pathogenic Microbiology. Aug. 2000. Dept. of Cell Bio.
& Molecular Genetics, UMD. 2 May 2006
<http://www.life.umd.edu/classroom/bsci424/Patho
genDescriptions/Corynebacterium.htm>.
Madigan, Michael T., and John M. Martinko. Brock
Biology of Microorganisms. 11th ed. London: Pearson
Prentice Hall, 2006. 386-387.
Sneath, Peter H., James T. Staley, and Stanley T.
Williams, eds. Bergey's Manual of Systematic
Bacteriology. Vol. 2. Baltimore: William and Wilkins,
1986. 1266-1280.