Examination of the prevalence of common nosocomial gastroenterological pathogens in

home and work kitchen settings

Derek Wiltz, Sally Moon

Our Lady of the Lake College

Introduction

Escherichia coli, Listeria monocytogenes, Salmonella, and Clostridium perfringens are some of the
most versatile and most common gastrointestinal related nosocomial pathogens. Some strains are important
members of the normal gut flora in human and animals, whereas others possess virulence factors that allow
them to cause infections in the intestinal tract or near other sites. Nosocomial infections are commonly
transmitted by health care providers and contaminated equipment. One of the main routes of transmission
are common vehicle transmission, where contaminated hosts can transmit to items such as food and water.
Furthermore, this transmission can result in food-borne illnesses, which are a major public health concern
worldwide. The World Health Organization defines food-borne disease as disease of infectious or toxic
nature caused by, or thought to be caused by, the consumption of food or water. Annually, an estimated 76
million illnesses, 325,000 hospitalizations, and 5,000 deaths are caused by food-borne diseases in the
United States (Powell 1121). Not only do these illnesses manifest at the workplace, but also at home. From
2009-2010, among the outbreaks with a known single setting where food was consumed, 21% were caused
by food consumed in a private home (Scallan 9).
This research aims of to target the bacteria Salmonella, L. monocytogenes, C.
perfringens and E. coli. The bacteria will be investigated by first identifying the
bacteria in samples gathered from food sources in a home and hospital followed by
analysis. It is hypothesized that samples obtained from the lounge of a hospital and
from a home will contain these bacteria that relates to sickness and that a higher
concentration of these pathogens are more frequent in the hospitals than in homes.
Materials and Methods
The samples were first chosen inside of a microwave door was obtained on August 31, 2015. This
was obtained from Burbank Commons, 4600 Burbank Drive Baton Rouge, LA 70820. The second and third
samples were obtained inside of a sink and inside of a refrigerator door on September 6, 2015 in the break
room of Tower Surgery at Our Lady of the Lake Regional Medical Center, 5000 Hennessy Blvd, Baton
Rouge, LA 70806. The fourth sample, a piece of meat from beef sample was from a refrigerator in a

residence obtained on August 31st, 2015. The samples were cultured in a Nutrient Agar plate and also stored
in a tube containing glycerin. Following storage, the cultured sample in the agar plate would be tested and
identified through a series of tests. Figure 1 shows a dichotomous flowchart below that identified each
sample through the process of elimination.
Figure 1
Dichotomous Flow Chart

After a week, the samples were transferred on a slide with an inoculation loop. Crystal violet was
added and stained over the fixed culture. Next, iodine was added on the smear, enough that it covered the
fixed culture. The slide was rinsed and counterstained with basic fuchsin solution for 40 to 60 seconds. The
slides were washed and blotted with bibulous paper to remove the excess water. Some of the bacteria
samples needed to be transferred onto new fresh plates to keep the bacteria viable. This was done by
transferring sample and isolation. A petri dish was divided into four equal quadrants. A loopful size was

lifted from the older plate and streaked slowly into the first quadrant. It is important that some of the
sample overlapped the second quadrant, second sample into third quadrant, etc. The previous samples that
were stored in glycerin was later used for DNA extraction and amplification. 1ml of sterile water was
pipeted into a 1.5 ml micro centrifuge tube. The tube was centrifuged for 10 min at 5000 x g (7500
rpm).The supernatant was removed completely and discarded, leaving a pellet. The bacterial pellet was
suspended in 180 l ATL Buffer and vortexed. 20 l proteinase K was added and mixed by vortexing, and
incubated at 56C for 10 minutes to lyse cells. The 1.5 ml micro centrifuge tube was centrifuged to remove
drops from the inside of the lid and 200l AL Buffer was added to the sample and incubated at 70C for 10
min.
Finally PCR Amplification was ran by first denaturation for 3 minutes at 98C, then denatured for
15 seconds at 95C. The primers that was used was the Universal for 16SrRNA (Nadkarni et al., 2002) and
it was annealed for 30 seconds on a temperature gradient (56.5, 58.4, 59.5 and 60C) and DNA was
extended for 1 minute at 70C. This was repeated steps for 40 cycles, with the final extension for 2 minutes
at 70C.
Results
Gram Staining Unknown Samples
All obtained samples were cultured and isolated, and a variety of tests were performed in
methodological order to accurately identify the microorganisms present. Gram staining, one of the key
differentiation methods necessary in the early stages of identification, was vital to determine the order of
corresponding steps to be followed. As seen below in Table 1, three out of four samples were Gram
positive, and the other was Gram negative. Additional differential tests were performed to not only identify
the organism present on the particular samples, but also to confirm the accuracy of the proposed
identification. The following were the results obtained from all tests performed that led to the identification
of the unknown organisms present on all four samples. Furthermore, Table 2 illustrates a lactose
fermentation test yielded a positive reading for the kitchen sink, microwave door, and meat, whereas the
refrigerator yielded a negative reading. Table 3 tested for the presence of catalase in the unknown
organisms whereas Table 4 provided the results of the starch hydrolysis test. Finally, as the organisms were

closer to being identified, Table 5 shows the results of the oxidase test and Table 6 yields growth results
from mannitol agar.
After these tests were complete picture 1 displays below a picture of Corynebacterium xerosis in
the refrigerator and meat sample, picture 2 reveals Veillonella meningitis in the kitchen sink sample, and
picture 3 illustrates Staphylococcus aureus in the microwave door sample.
Table 1
Gram Staining Results

SAMPLE

GRAM STAIN RESULT

Refrigerator

Gram Positive (+), Bacilli

Kitchen Sink

Gram Negative (-), Cocci

Microwave Door

Gram Positive (+), Cocci

Meat

Gram Positive (+), Cocci

A gram stain was performed on all unknown samples, and the resulting color and morphology are
presented.

Table 2
Lactose Fermentation Test Results
Refrigerator

Negative (-)

Kitchen Sink

Positive (+)

Microwave Door

Positive (+)

Meat

Positive (+)

The unknown organisms ability/disability to ferment lactose was tested by using glucose agar in Durham
tubes and confirmed with the presence/absence of growth on MacConkey agar.

Table 3
Catalase Test Results
Refrigerator

Positive (+)

Kitchen Sink

Positive (+)

Microwave Door

Positive (+)

Meat

Positive (+)

Hydrogen peroxide was used to observe bubble production on the corresponding samples more than once,
to identify the organisms that produce catalase.

Table 4
Starch Hydrolysis Test Results
Refrigerator

Negative (-)

Meat

Negative (-)

Only the organisms questionable for its ability to break down starch were used for the above test, which
confirmed that neither sample had Bacillus species.

Table 5
Oxidase Test Results
Kitchen Sink

Negative (-)

The oxidase test was performed and analyzed by the resulting color of the tested colony.

Table 6
Mannitol Salt Agar Growth Results
Microwave Door

Yellow Growth; Positive (+)

Mannitol salt agar was used to differentiate between Staphylococcus and Streptococcus species, by
observing the change in color of the media due to the altered acidity of the media, which resulted from the
fermentation byproducts.

Table 7
Identifying Unknown Organisms
SAMPLE

IDENTIFIED UNKNOWN ORGANISM

Refrigerator

Corynebacterium xerosis

Kitchen Sink

Veillonella meningitis

Microwave Door

Staphylococcus aureus

Meat

Corynebacterium xerosis

The final three tests were used as the final determinant differential test to identify and confirm the four
unknown organisms present on the samples.
Picture 1
C. xerosis from Refrigerator and Meat Sample

The refrigerator tested for C. xerosis

Source: http://www.microbelibrary.org/component/resource/gram-stain/2864-gram-stain-gram-positiverods

Picture 2
V. meningitis from Kitchen Sink

Source: http://microblog.me.uk/359

Picture 3
S. aureus from Microwave Door

Source: http://www.bacteriainphotos.com/bacteria%20under%20microscope/staphylococcus%20aureus
%20microscopy.html
PCR Amplification Results
The known samples identified in the previous test were then sent for analysis by PCR
Amplification. Figure 1 displays the melting curve for all of the tested samples. The graph results is ideal,
showing that the samples did eventually melt at a reasonable temperature to undergo isolation. Figure 2
displays the amplification peaks. Again this is a very good result because it shows that the samples
underwent an ideal amplification and was also able to undergo many cycle, which yielded very high
amounts. And finally Figure 3 shows the data translated onto a Microsoft Excel Spreadsheet.

Figure 1
Melting Curve Analysis

The melting curve is a curve that tells what temperature the samples must be near for optimal annealing.
Most samples peaked near identical temperatures.

Figure 2
Peak Amplification Results

This figure shows an artificial increase in the number of copies of a particular DNA fragment into millions
of copies through replication of the segment into which it has been cloned.

This figure displays all of the data from the PCR, including annealing temperature, melting temperature
and species ID.
Discussion
The results were inconsistent, because only one species was identified positively, which was the
Methicillin-resistant Staphylococcus aureus (MRSA). The samples during amplification did not fluoresce
properly, which left the species identification unknown. Also, the S. aureus and E. coli colony counts were
inconsistent to the count of PCR. This left the samples very inconclusive to be compared to any known
bacteria with identical count numbers. On the other hand, many of the mean counts for each sample were
identical to each other, which shows that at least the unknown bacteria was consistent. For example, the
microwave door and meat shared one mean count, while as the kitchen sink and refrigerator door shared
two mean counts. This means that the microwave door and meat still has only one bacteria, while the
kitchen sink and refrigerator doors has at least two. Although, the data showed inconclusive stats to
compare which sample had which bacteria and at a higher prevalence, does this mean more bacteria is
becoming resistant? Also, what does this mean overall for food safety in the home and workplace? How
can PCR improve to detect pathogens? And for future studies, do we need to improve pathogen prevention
or better treatment?
Overall, the PCR is the most sensitive of methods to detect microbial pathogens in specimens. In
particular, when specific pathogens that are difficult to culture in vitro or require a long cultivation period
are expected to be present in specimens, the value of PCR is very significant. However, it is important that
the sensitivity and specificity of a PCR assay is dependent on target genes, primer sequences, PCR
techniques, DNA extraction procedures, and PCR product detection methods to be reliable.

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