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 illnessSince the beginning of human history, food borne illness has been an issueVarious 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: dryingfreezingfreeze-dryingsaltingcuringcanningpicklingirradiationtreating with sugartreating with inert gases (such as carbon dioxide)

S

Transient flora found on human skin and hands plays an important role in food contaminationWhen those involved in food preparation fail to observe hygienic methods

History of food borne illness (cont.)

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 testedHypothesis

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 MethodsOverall 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 consideredAgar 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 determinedIt 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 requiredBoth saline wetted and dry sterile cotton swabs were considered:Wet cotton swabs were determined to be most effective for obtaining the largest sample of microorganismsMicroorganisms adhere more readily to wet surfacesA 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 organismsThe 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 MethodsDay 1Made Mannitol Salt AgarAutoclaved and cooled mediumDay 2Poured Mannitol Salt Agar platesDay 3Obtained Trypticase Soy Agar (TSA) platesObtained a package of sterile cotton swabs, and a tube of diluted salineSwabbed 7 objects:Bathroom sink faucet (1st floor)Bathroom (interior) doorknob (1st floor)Micro bench sink faucet (Room x)Front door handleMicro buildingHandicapped door buttonMicro buildingComputer mouse in labStair rail (1st floor)

Detailed Methods (cont.)

Day 3 (cont.)Swabbing method used:Dipped tip of sterile swab into salineSwabbed 1 of 7 objectsStruck inoculated swab on sterile agar plate with zigzag motionA new swab was used for each plate4 plates (2 TSA and 2 Mannitol Salt Agar) were used for each objectDiscarded the swabIncubated all plates at 37C for 48 hours

Detailed Methods (cont.)

Day 4Observed growth of different colony types on TSA platesObserved growth of colonies on Mannitol Salt Agar platesObserved fermentationPhotographed certain colonized platesCounted and recorded different types of coloniesSelected different types of colonies from each Mannitol Salt Agar plateStruck the selected colonies on (new) Mannitol Salt Agar plates, on (new) TSA plates, and on (new) Blood Agar platesIncubated all plates at 37C for 24 hours

Detailed Methods (cont.)Day 5Observed Mannitol Salt Agar plates Observed fermentationObserved TSA platesObserved Blood Agar platesObserved hemolysisPerformed catalase assay on pure colonies isolated from Mannitol Salt Agar platesPerformed gram stain on pure colonies isolated from Mannitol Salt Agar platesPerformed CoAgulase assay on certain pure colonies isolated from Mannitol Salt Agar platesBactistaph latex 150 testPhotographed relevant resultsDay 6Observed results from CoAgulase assay

Detailed Rationale Behind MethodsDay 3Saline was used: To maintain highest degree of sterilitySince bacteria adhere more readily to wet surfaces than to dry surfacesIn a diluted form so growth of non-salt tolerant organisms was uninhibitedA new swab was used for each plate to reduce the risk of contamination by organisms not found on objects of interestTSA 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 growAll plates were incubated at 37C for 48 hours:37C is in the optimal growth temperature range for organisms that could be found on objects of interest48 hours is in the optimal growth time range for organisms that could be found on objects of interest4 plates (2 TSA and 2 Mannitol Salt Agar) were used:To safeguard from errorIf 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 4Different 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 plateStruck 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 aureusStaphylococcus 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 aureusStaphylococcus aureus has -hemolysis on Blood Agar

Detailed Rationale (cont.)

Day 5Catalase test was performed to:Confirm that cultures from Mannitol Salt Agar plates were Staphylococci (positive) not Streptococci (negative) Indicating that cultures were Staphylococcus aureusGram stain was performed to:Confirm that cultures from Mannitol Salt Agar plates were gram positive not gram negativeIndicating that cultures were Staphylococcus aureusConfirm that bacteria colonized on Mannitol Salt Agar plates formed clusters not chainsIndicating 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 agglutinationIndicating that the cultures were Staphylococcus aureusCoAgulase assay was performed to:Confirm that isolated colonies were CoAgulase positive not CoAgulase negativeIndicating that the cultures were Staphylococcus aureus

Expected ResultsIt 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

ResultsFindings

Summary of Findings

Relationship of findings to expectations

FindingsTSA plate 1 was inoculated with microorganisms from the bathroom sink faucet:7 small white colonies grew8 large white colonies grew

TSA plate 2 was inoculated with microorganisms from the bathroom sink faucet:16 small white colonies grew31 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 grew1 medium white colonies grew

Mannitol Salt Agar plate 1 was inoculated with microorganisms from the computer mouse:2 small white colonies grew3 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 grew1 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 grew1 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 grew1 small white colony grew1 medium white colony grew

TSA plate 2 was inoculated with microorganisms from the bathroom door knob:7 small white colonies grew9 large white colonies grew

Mannitol Salt Agar plate 1 was inoculated with microorganisms from the bathroom door knob:2 medium colonies with yellow zones grew2 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 plateGram positive rodsSnapping replication was observedCatalase positive

A Mannitol Salt Agar plate was inoculated with microorganisms from the bathroom sink:Growth and fermentation on Mannitol plateGram positive cocci organized in grape like clustersCatalase positive

Findings (cont.)A Mannitol Salt Agar plate was inoculated with microorganisms from the stair rail:Growth and fermentation on Mannitol plateGram positive rods Snapping replication was observedCatalase negative

A Mannitol Salt Agar plate was inoculated with microorganisms from the computer mouse:Growth and fermentation on Mannitol plateGram positive rods Snapping replication was observedCatalase positive

Findings (cont.)A Blood Agar plate was inoculated with microorganisms from the bathroom door handle:Growth on Blood Agar-hemolysis

A Blood Agar plate was inoculated with microorganisms from the bathroom sink:Growth on Blood Agar -hemolysis

A Blood Agar plate was inoculated with microorganisms from the stair rail:Growth on Blood Agar-hemolysis

A Blood Agar plate was inoculated with microorganisms from the computer mouse:Growth on Blood Agar-hemolysis

Summary of Findings Microorganisms isolated from the objects exhibited a variety of phenotypes when grown on TSA plates:Small white bacterial coloniesSmall yellow bacterial coloniesMedium white bacterial coloniesLarge white bacterial coloniesThe phenotypes above indicate that a very diverse bacterial population is present on the tested objectsThis data supports the first hypothesis that numerous types of microorganisms would live on tested objects

Summary of Findings (cont.) Microorganisms isolated from the:Stair railComputer mouseBathroom door handleAre suspected to be Cornyebacteria since:They undergo snapping replicationForm Chinese lettersAre gram positive rodsThis 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 clustersAre catalase and CoAgulase positiveHave -hemolysisFerment MannitolThese phenotypes are identical to those exhibited by Staphylococcus aureusThis 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 aureusWhy Staphylococcus aureus was expected to be foundIncidence of disease Virulence of Staphylococcus aureusComments on protocolComments on resultsComments on overall significance of project

Description of Staphylococcus aureusPhenotypeGram positive cocciFound in pairs, chains, or grapelike-clustersProduces heat stable enterotoxinSalt tolerantFerments MannitolCatalase positiveCoAgulase Positive

Description of Staphylococcus aureus (cont.)HabitatAirDustSewageEnvironmental surfaces50% of humans (higher in health professionals)FoodsPoultry and egg productsEgg, tuna, potato, and chicken saladsMilk 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 probableStaphylococcus aureus is a component of the normal flora for 50% of all peopleCarriers of Staphylococcus aureus transfer the bacteria to every touched object Regardless of hand washingIn proper growth conditions the bacteria will propagate on objects such as those testedAs Staphylococcus aureus contaminated objects are used, bacteria are transferred to a users transient floraStaphylococcus aureus was isolated on tested objectsThus, hand washing does not preclude the transfer of Staphylococcus aureus to food

Incidence of diseaseThe incidence of illness is difficult to pinpoint due to a number of reasonsPoor response from victims during interviewsMisdiagnosis (symptoms are very similar to those of Bacillus cereus toxin)Inadequate collection of lab samplesImproper lab examination

Virulence of Staphylococcus aureusCauses staphyloenterotoxicosis (food poisoning)Operates by toxin, not colonizationID50 1.0 microgramOnset of illness is rapid and varies based on:Amount of food ingestedAmount of toxin present in foodIndividual susceptibilityGeneral health of victim

Virulence of Staphylococcus aureus (cont.)Symptoms:NauseaVomitingAbdominal crampingHeadacheChanges in pressure and blood ratesAverage recovery time for mild case: 2-3 days

Virulence of Staphylococcus aureus (cont.)

TreatmentRestFluidsMedicines to calm stomachHospitalization/Intravenous therapy for more severe cases (usually elderly or infants)

Virulence of Staphylococcus aureus (cont.)

PreventionWash hands and under nails vigorously and oftenDo not prepare food with an eye or nose infectionDo not serve food to others with open woundsKeep food preparation areas sanitaryStore cooked food in wide, shallow containers and refrigerateDo 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 resultsSaline wetted swab and streak inoculation method was successful:Isolated colony growth occurredTesting and differential media:CatalaseCoAgulaseMannitol Blood

Comments on ResultsComments on ResultsFound numerous colonies resembling Corynebacteria, a Gram-Positive rod generally found in soilMethylene blue staining did not reveal metachromatic granules, however not all species exhibit this traitThis finding is consistent with concept of bacteria found on unwashed handsUnable 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 projectThis 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

CDC. Outbreak of community-associated methicillin-resistant Staphylococcus aureus skin infections---Los Angeles County, California, 2002--2003. Morbidity and Mortality Weekly Report 2006;52:88.

U.S. Food and Drug Administration. Staphylococcus aureus. 1992. Food borne Pathogenic Microorganisms and Natural Toxins Handbook.

ReferencesRollins, David M. "Corynebacterium Summary." BSCI 424 Pathogenic Microbiology. Aug. 2000. Dept. of Cell Bio. & Molecular Genetics, UMD. 2 May 2006 .

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.