MOLECULAR DETECTION OF FECAL-BACTERIAL CONTAMINATION ON
GROCERY STORE SHOPPING CART HANDLES WITHIN THE SACRAMENTO
REGION
Melissa Ann Morris
B.S., California State University, Sacramento, 2003
THESIS
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
BIOLOGICAL SCIENCES
(Molecular and Cellular Biology)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
FALL
2010
MOLECULAR DETECTION OF FECAL-BACTERIAL CONTAMINATION ON
GROCERY STORE SHOPPING CART HANDLES WITHIN THE SACRAMENTO
REGION
A Thesis
by
Melissa A. Morris
Approved by:
______________________________, Committee Chair
Susanne W. Lindgren, Ph.D.
______________________________, Second Reader
Thomas R. Peavy, Ph.D.
______________________________, Third Reader
Enid T. Gonzalez, Ph.D.
________________________
Date
ii
Student: Melissa A. Morris
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.
___________________________, Department Chair
Rose Leigh Vines, Ph.D.
Department of Biological Sciences
iii
_____________________
Date
Abstract
of
MOLECULAR DETECTION OF FECAL-BACTERIAL CONTAMINATION ON
GROCERY STORE SHOPPING CART HANDLES WITHIN THE SACRAMENTO
REGION
by
Melissa A. Morris
Shopping cart handles have recently been targeted by the popular press as a
highly contaminated public surface. Both retailers and consumers are concerned due to
the heavy utilization of the grocery industry within the United States. Recent
epidemiological investigations have implicated riding in shopping carts as risk factors for
food-borne pathogen infection in infants. Studies have also revealed the existence of
food-borne pathogens on the surface of food and packaging as well as crosscontamination from surfaces and hands that have not been effectively cleaned.
Additionally, transfer of fecal-borne bacteria from fomite surfaces to hands has been well
studied. Taken together, data from all of these studies suggest that a shopper could
acquire pathogens from contact with grocery shopping cart handles that have been
contaminated.
Although inferences have been drawn, no studies to date have been published that
have specifically investigated the type and levels of fecal-borne and food-borne bacteria
present on grocery shopping cart handles. The purpose of this study was to fill this
informational gap by evaluating the prevalence of fecal-bacterial and food-borne
iv
pathogen contamination on local shopping cart handles utilizing a combination of
molecular methods and traditional cultivation techniques. Additionally, we evaluated if
there was a difference in the prevalence of bacteria between traditional grocery
establishments and discount/outlet type grocery establishments. Specifically, this study
investigated the total aerobic bacterial populations present, as well as identified the
presence of Escherichia coli spp., Salmonella spp., and Shiga toxin producing
Escherichia coli (STEC) on shopping cart handles from retail grocery stores in the
Sacramento Region. A multiplex PCR method was successfully developed to detect the
presence of STEC and Salmonella from a mixed culture. This method was used in
tandem with PCR to identify generic Escherichia coli as an indicator of fecal
contamination. These methods were applied in a survey of 12 stores in three regional
locations east of Sacramento. Between March and October 2009, each store was visited
five times and ten carts were selected at random and sampled at each visit. In total, 600
bacterial samples were successfully collected and analyzed utilizing PCR, multiplex
PCR, and traditional cultivation for this study.
Out of 600 samples, one sample (0.17%) tested positive for STEC’s stx1 and one
sample tested positive for Salmonella spp. (0.17%). For the fecal contamination indicator
test, 582 were found to be positive for E.coli spp. (97%). The total number of aerobic
bacteria found on the cart handles varied from 0 to over 53,000 colony forming units.
Finally, comparisons of the means of total aerobic counts were made between regional
locations and grocery establishment type using a 2-way ANOVA test. Statistical analysis
of the mean total aerobic counts revealed no significant difference between regional
v
locations. There was also no statistically significant difference between the two grocery
establishment-types tested (P > 0.05).
____________________________________ Committee Chair
Susanne W. Lindgren, Ph.D.
________________________
Date
vi
ACKNOWLEDGEMENTS
I dedicate this thesis to my husband, Ronald, and my children, Lilyann and
Charles. Thank you for your many sacrifices and endless love and support.
I would also like to thank Dr. Susanne Lindgren and Dr. Robert Metcalf for your
invaluable support and input. Special thanks to Dr. Dennis Huff for making me snort
with laughter and fall in love with microbiology.
vii
TABLE OF CONTENTS
Page
Acknowledgements ........................................................................................................... vii
List of Tables ..................................................................................................................... ix
List of Figures ..................................................................................................................... x
Introduction ....................................................................................................................... 11
Materials and Methods ...................................................................................................... 21
Results ............................................................................................................................... 44
Discussion ......................................................................................................................... 60
Appendix ........................................................................................................................... 69
Literature Cited ................................................................................................................. 74
viii
LIST OF TABLES
Page
Table 1. PCR Primers Used in this Study ......................................................................... 24
Table 2. Regional Location Demographics ...................................................................... 33
Table 3. Store Information, Number of Carts Sampled and Sampling Schedule ............. 35
Table 4. Relative Mean CFU Collected per Cart and Standard Deviations of Compared
Sample Groups ................................................................................................... 54
Table 5. Results of 2-way ANOVA .................................................................................. 56
Table 6. Petrifilm™ E. coli, Total Coliform Counts and PCR Detection Results of
Samples Collected 9/22/2009 ............................................................................ 59
ix
LIST OF FIGURES
Page
Figure 1. Sensitivity and Method Development Assay .................................................... 31
Figure 2. Map of Regional Locations Selected for Grocery Shopping Cart Sampling .... 33
Figure 3. Sample Collection Procedure. ........................................................................... 38
Figure 4. Sample Processing Procedure............................................................................ 41
Figure 5. Five-plex PCR Alignment with Complete and Separate Milieu ....................... 47
Figure 6. Dilutions of Complete Milieu and Five-Plex PCR Alignment .......................... 47
Figure 7. E.coli 16S PCR Product Alignment .................................................................. 48
Figure 8. Sensistivity Assay for Multiplex PCR Non-Specific Enrichment Method ....... 50
Figure 9. Sensitivity Assay for E. coli spp. 16S Non-Specific Enrichment Method ........ 50
Figure 10. Preliminary Sample Multiplex PCR Results. .................................................. 52
Figure 11. Preliminary Sample 16S PCR Results............................................................. 52
Figure 12. Combined Mean Total Aerobic Bacteria Count, Grouped by Establishment
Type, Chains, and Regional Location ............................................................. 56
Figure 13. Mean Total Aerobic Bacteria Counts per Store, Grouped by Establishment
Type and Regional Location.. ......................................................................... 57
x
11
INTRODUCTION
The grocery store industry is one of the largest and most prevalent industries in
the United States. Recent investigations have not only identified shopping cart handles as
one of the most biologically contaminated public surfaces, but also have implicated
riding in shopping carts as a risk factor for food-borne pathogen infection in infants (21,
27). Taken together, these findings suggest that a grocery shopping cart handle may
serve as a transmission source for pathogenic organisms to susceptible individuals.
However, no studies to date have attempted to support this hypothesis or to identify the
presence of specific bacteria on these surfaces.
Pathogen transmission occurs by either direct or indirect pathways. Direct
pathways include the human-to-human transmissions, such as the transmission of HIV
through contact with contaminated body fluids (CDC. www.cdc.gov/hiv June 8th 2010).
Indirect transmission occurs when a non-living agent is involved in the transfer of the
pathogen to a susceptible individual such as through air, food, water and inanimate
objects known as fomites. Transmission by air, food, and water has been well
documented for many pathogens such as Listeria monocytogenes through the
consumption of contaminated deli meat (1). However, only recently have studies begun
to understand fomites as transmission vectors for human pathogens (6, 8, 16, 51).
Various factors are thought to play a role in potential disease transmission from
contact with contaminated fomites. These factors include the type of fomite surface, the
pathogen’s survivability and concentration on the fomite, as well as the type of physical
contact an individual has with the fomite and the immunological health of the individual
12
(6, 9, 39, 40). Researchers have determined that the potential transfer rates of bacteria
from non-porous contaminated surfaces to human hands can be as high as 66% (41). In
addition, the transfer of bacteria to other skin surfaces, such as from the fingertips to the
mouth-lip area can occur at a rate of 30-40% (41). The virulence of microbes is another
key factor in fomite transmission. Some microbes require high doses to cause infection in
healthy individuals, while others may cause disease by contact with only a few cells (6,
8). For example, it is thought that Staphylococcus aureus requires 106 colony forming
units (CFU) to cause an infection in skin, however ingestion of between one and six CFU
have been calculated in cases of Salmonella typhimurium infection (6).
Recent epidemiological studies have implicated fomites in the transmission of
human pathogens within high-exposure, contained environments such as hospitals, childcare facilities, long-term care facilities, and sports facilities (6, 8, 16, 29, 32, 33, 51).
Organisms implicated in fomite-transmission in these environments include rotavirus,
rhinovirus, methicillin-resistant Staphylococcus aureus (MRSA), and Serratia marcesens.
These organisms are responsible for gastrointestinal illness, the common cold,
necrotizing fasciitis, and catheter associated bacteremia, respectively (8, 16, 32, 33).
Evidence has also implicated the presence of specific food-borne pathogens on surfaces
within the immediate environment of infected individuals. A review article in 1997, by
Bloomfield et al., cited nine epidemiological reports that implicated contaminated
surfaces such as equipment, cutting boards and other kitchen surfaces within high
exposure environments in the potential infectious transmission of the food-borne
pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Shigella
13
sonnei (6). These epidemiological studies illustrate the survivability and potential
transfer of traditionally food-borne pathogens from fomites, but fail to address the risk of
exposure with surfaces in more transient environments such as grocery stores.
A study in 2005 by Reynolds et al., evaluated the presence of biological
contamination on numerous environmental surfaces by measuring protein and
biochemical markers for human body fluids such as blood, saliva, urine and sweat, as
well as fecal and total coliform bacteria (40). Their results indicated that shopping carts
handles ranked third, amongst the surfaces sampled, for biological contamination. In
2006, an epidemiological study by Jones et al., identified riding in shopping carts near
meat or poultry products as an associated risk factor in infant Salmonellosis (27). This
same research group conducted an additional study a year later that identified shopping
carts as an associated risk factor for infant Campylobacteriosis (21). The combined
results of these studies suggest that shopping carts may, in-fact, play a role in the
transmission of enteric pathogens.
A logical source of contamination of grocery store shopping carts would be from
the consumer’s hands. The presence of enteric bacteria on hands and surfaces has been
well studied within high-exposure environments such as medical and child care
communities (6, 8, 16, 29, 32, 33, 51). One such study found that 30-56% of children in
child-care facilities had fecal coliforms on their hands (33). Recently, a study
investigated the presence of fecal-bacteria on the hands of adults using the public transit
system. This study, conducted in five United Kingdom cities, found that 28% of the
commuters sampled were found to have bacteria of fecal origin on their hands (28).
14
What is important about this study is that it attempted to characterize bacterial
contamination on people’s hands within a more transient environment similar to that of
grocery stores. Taken together, the high prevalence of fecal bacteria present on people’s
hands revealed by the UK study, highly suggests that a customer's hands could be a
source of contamination for grocery shopping cart handles.
Another source for bacteria on shopping cart handles could potentially be from
the raw foods and packaging that are handled in the grocery store and placed within the
cart. Numerous studies have investigated and confirmed the presence of food-borne
pathogens in or on food products (4, 5, 11, 20, 25, 36, 38, 39, 41, 43). For example, a
study within our laboratory detected Shiga toxin producing Escherichia coli within 11%
of local ground beef samples (36). In addition, studies have confirmed and measured the
presence of specific food-borne pathogens Campylobacter spp., Escherichia coli spp.,
and Salmonella spp. on the external packaging of raw meat (9, 24). Due to the potential
of these organisms to be present on food surfaces and packaging, and the associated risk
factors for food-borne pathogens Salmonella spp. and Campylobacter jejuni to cause
illness in infants from riding in shopping carts, it seems likely that these pathogens could
also be present on the surface of grocery shopping cart handles (22, 27).
In this study, we attempted to address this information gap by evaluating the
prevalence of several well characterized enteric bacteria. Organisms evaluated within
this study included normal enteric Escherichia coli (E. coli) as well as two specific foodborne pathogens: Shiga toxin-producing E. coli (STEC) and Salmonella. E. coli colonize
the intestinal tracts of all warm-blooded animals. Most strains of E.coli are non-
15
pathogenic and serve as part of the normal flora of the digestive system. Their presence
benefits the host in multiple ways including the production of vitamin K and prevention
of pathogenic organism growth and establishment through competitive inhibition (17). E.
coli can be shed through feces and survive outside of the body. Because of this ability, E.
coli is often used as an indicator of fecal contamination. Evidence of fecal contamination
by identifying this indicator species suggests the presence of other disease-causing
organisms found in feces such as STEC and Salmonella. According to the United States
Centers for Disease Control (CDC), STEC and Salmonella combined are responsible for
over 1.5 million cases of food-borne illness each year (CDC www.cdc.gov June 8th
2010).
STEC is reported in an estimated 70,000 cases of illness in the United States each
year (CDC. www.cdc.gov/ecoli/ June 8th 2010). This number is thought to be a low
estimate as individuals often do not seek care for diarrheal illness and do not have
specific testing done. Many labs only test for E. coli O157:H7, which is a subset of the
STEC group. Symptoms of STEC infection include diarrhea with or without blood,
severe stomach cramps, and vomiting. In an estimated 10% of cases, the infection may
lead to the more severe and life-threatening complication called hemolytic uremic
syndrome (HUS) (CDC. www.cdc.gov/ecoli/ June 8th 2010). HUS is characterized by
hemolytic anemia and renal damage which can lead to life long renal complications
and/or death. HUS is primarily observed in children, elderly, and individuals with
weakened immune systems.
16
The main causative agent for HUS are the Shiga toxins that STEC produce.
There are two types of Shiga toxin, Shiga toxin 1 (Stx1) coded by the stx1 gene and Shiga
Toxin 2 (Stx2) coded by the stx2 gene. Both Shiga toxin subtypes are composed of two
subunits, A and B. The B subunit binds to Gb3 glycolipids found on the surface of
susceptible cells. After the toxin binds to a cell and is internalized, the A subunit is
enzymatically activated and inhibits cellular protein synthesis and the cell dies.
Interestingly, Gb3 is found in high concentration on the surface of renal cells in young
children and seniors, which explains why renal toxicity is especially high in these
individuals (15, 17). STEC strains can carry either stx1 or stx2 or both. Stx2 has been
found to be more potent toxin and more likely to lead to HUS (15, 17). The most
commonly identified member of the STEC group in the United States is E.coli O157:H7.
However, as methodology improves for the detection of Shiga toxins, other serotypes
such as O111, O91 and O126 have been implicated in disease (3, 15, 17, 23).
Ruminant animals, such as cattle and sheep serve as primary reservoirs for STEC
(3, 4, 5, 12). STEC may be transmitted to humans by contact with these animals, or their
waste, or by consuming contaminated food or water. Foods that have been implicated in
STEC outbreaks include raw or undercooked beef, un-pasteurized milk, un-pasteurized
apple cider and juice, as well as fresh produce such as spinach, cantaloupe, alfalfa and
radish sprouts (12, 23). At a more local level, studies within our research group have
indicated that STEC are present in 11% of ground beef obtained grocery stores in the
regional Sacramento area (36).
17
Salmonella is responsible for an estimated 40,000 confirmed cases of food-borne
illness and 400 deaths each year in the United States (CDC. www.cdc.gov/salmonella/.
June 8th 2010). Common foods where this pathogen has been found include beef,
poultry, milk and eggs. It has been estimated that 29% of raw chicken found in grocery
stores is contaminated with Salmonella (24). Because the symptoms of Salmonella
infection are less severe in comparison to STEC, the actual number of cases is thought to
be closer 1.4 million cases per year. S. enteritidis, S. typhimurium, and S. newport are the
most commonly cultured isolates from patients within the U.S. (CDC.
www.cdc.gov/salmonella/. June 8th 2010). Symptoms of Salmonella infection include
diarrhea, fever, and abdominal cramps. Most infected persons recover completely
without treatment. However, a small percentage of people may have long lasting
sequelae, known as Reiter’s syndrome, regardless of antibiotic treatment. Reiter’s
syndrome is also known as “reactive arthritis”. This disease can affect multiple areas of
the body and can be self limiting, recurrent, or progressive and can lead to life-long
chronic arthritis (CDC. www.cdc.gov/salmonella/. June 8th 2010). During Salmonella
infection, some patients require hospitalization for dehydration or septicemia when the
organism travels into the blood stream and other organs and it becomes life-threatening.
Septicemia is fatal without treatment.
An essential virulence mechanism for Salmonella is its ability to invade the host
intestinal epithelium. Salmonella is capable of inducing the host cell to modify its actin
filament arrangement to engulf the bacteria. This action is called “ruffling” due to its
appearance under the microscope. Ruffling is under the control of a large group of
18
molecules controlled for by a series of genes located on the Salmonella pathogenicity
island I (SPI 1) including a group of genes called inv (invA-D). The invA gene is
required for assembly of a type III secretion system T3SS. T3SS are integral to
Salmonella invasion, virulence, and are involved in secreting effector molecules that
induce host cell ruffling and bacterial entry (13). Specifically, invA codes for a 71 kilo
Dalton (kDa) putative inner membrane protein that appears to be homologous with many
other type III secretion proteins. Mutations within the inv group, depending on the
location of the mutation, either reduce or completely abrogate the ability of Salmonella to
invade intestinal epithelial cells (13).
Traditional methods for detection of STEC and Salmonella in food have included
culturing the food item on selective media followed by characterization of suspect
colonies with additional biochemical tests and immunoassays. In general, this process
requires multiple days for successful identification of the pathogen. To overcome the
protracted nature of traditional detection methods, and to enhance the sensitivity and
specificity of detection, a number of molecular diagnostic methods have been developed,
including methods that utilize Polymerase Chain Reaction (PCR).
PCR is a technique for amplifying specific regions of DNA by the use of
sequence-specific primers to serve as specific points of attachment for DNA polymerase.
A PCR reaction is carried out by manipulating the reaction temperature to facilitate DNA
dissociation, annealing, and elongation steps that normally occur in replication. The
precise manipulation of temperature during PCR allows the process of DNA replication
to occur outside of normal intra-cellular induced environment. This technique allows
19
researchers to identify the presence of a specific sequence against a background of mixed
genomic DNA. The general technique of PCR can be modified to target several unique
sequences within a single reaction. This technique is called multiplex PCR. When
applied to environmental surfaces, the use of multiplex PCR thus allows for the
identification of multiple pathogens from a single swab sample. Several studies,
including those within our research laboratory, have confirmed the use of Multiplex PCR
to identify individual or multiple pathogens within food, environmental matrices, and
surface swabs using gene-specific primers (2, 11, 20, 36, 38, 42, 43, 45, 49).
There are no published studies to date that have attempted to utilize multiplex
PCR to investigate fecal-bacterial contamination on grocery shopping cart handles
specifically. With the recent implication of shopping carts in the transmission of enteric
pathogens to susceptible individuals, and the identification of these surfaces as highly
contaminated, the next logical step would be to identify and characterize fecal-bacterial
contamination on grocery shopping cart handles. The application of multiplex PCR in
this capacity would allow for rapid and accurate identification.
The intent of this study was to evaluate fecal-bacterial contamination on grocery
shopping cart handles by determining the prevalence of contamination on this fomite.
The study was designed to incorporate grocery stores in three different socio-economic
regional locations in the Sacramento area. A survey of 600 shopping carts in three
regional locations east of Sacramento was completed between March and October 2009.
A total of 12 stores were visited five times each, and ten carts were sampled per visit.
The first goal of this study was to determine the prevalence of bacterial contamination on
20
shopping cart handles within the Sacramento region. I hypothesized that carts within the
study area would reveal high populations of total aerobic bacteria (>106 cfu).
Additionally, I hypothesized that I would detect the presence of E. coli spp., STEC and
Salmonella spp. in a low percentage of carts sampled (<5% of carts). The second goal of
this study was to compare the contrasting grocery establishment types. The survey of
grocery store carts was also designed to include four different grocery store chains. Two
of the chains sampled represented traditional grocery establishments and two represented
discount/outlet type grocery establishments. While the null hypothesis is there would be
no difference between traditional and discount/outlet grocery establishments, public
perceptions on the demographics and general cleanliness of the shopping environment
suggests otherwise.
Our study applied multiplex PCR to identify the presence of Salmonella spp.,
STEC, and E. coli spp. Both Salmonella spp. and STEC were targeted for identification
by their primary virulence factors which clearly distinguish them from all other
organisms. The invA gene that controls Salmonella’s signature ruffling action for
intracellular invasion is highly conserved and served as target for PCR (13, 20, 22).
Likewise, Shiga toxin 1 (Stx1) coded by the stx1 gene and Shiga toxin 2 (Stx2) coded by
the stx2 gene were utilized to target STEC for identification (20, 35). Additionally, a
single PCR reaction assay was used to amplify the highly conserved16S ribosomal
subunit gene within E. coli spp. for use as an indicator of fecal bacterial contamination
(49). Finally, traditional plating techniques were used to evaluate the total levels of
aerobic bacterial contamination.
21
MATERIALS AND METHODS
Multiplex PCR Primer Design
An initial multiplex PCR method was developed to detect the presence of four
different diarrheal disease-causing bacterial pathogens in a mixed culture. These four
enteric pathogens included: STEC, Campylobacter jejuni, Listeria monocytogenes, and
Salmonella spp.. Our lab has successfully utilized the methodology and primers
developed by Meng et al. 2002 for the detection of STEC by targeting genes stx1 and stx2
(35). When designing new primers, we used these primers as a foundation for our study,
and designed additional primers to add to the stx primers in order to identify the
additional pathogens. Studies evaluating the function of genes of these three pathogens
were used to create a list of potential gene targets for PCR amplification. To evaluate the
uniqueness of each gene, complete gene sequence information was obtained from the
National Center for Biotechnology Information (NCBI ) website nucleotide search engine
(http://www.ncbi.nlm.nih.gov/nucleotide/). The nucleotide sequences were entered into
the NCBI Basic Local Alignment Search Tool (BLAST) program for comparison against
all sequences within the database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). This
comparison allowed the evaluation of potential homology of the sequence with sequences
of various strains of the same organism as well with other organisms that may cause false
positives within the reaction. The pool of potential sequences was narrowed down as
potential PCR targets based on what role the gene played in pathogenicity, how highly
conserved and unique to the organism the sequences were. Three genes were selected
22
using this criteria; hlyA of L. monocytogenes, mapA of C. jejuni, and invA of Salmonella
spp.. The design of each set of primers incorporated three important aspects: 1) to detect
a specific gene unique to the organism of interest, 2) to not cross react or inhibit the
ability of the other primers in solution to bind to their intended sequence, and 3) produce
fragment sizes within 100-1000bp in a Multiplex PCR that would be distinguishable from
the other PCR products on an agarose gel when analyzed by electrophoresis. Once target
genes were selected, potential primers were then created and screened using stringent
criteria. Probes selected for multiplexing would need to be between18 and 24 base pairs
(bp) in length. Using the empirical equation to calculate primer melting temperatures
(Tm°C = 4(G + C) + 2(A + T)), we screened each potential primer for a Tm that would
be within the range of the existing primers (Stx1 = 59.6 – 60.4 °C, Stx2= 57.7 – 59.6 °C).
To also meet this requirement and maintain efficient melting and annealing, we required
primers to be between and a GC content of at least 50% to 60%. To avoid 3’ clamp
formation and non-specific interactions, the 3’ end was to be composed of a G, C, CG, or
GC and no sequential runs of three Cs or Gs were allowed. Primers were also evaluated
to ensure that they did not form hairpin structures, primer-dimers, and other non-specific
interactions when mixed together with the other primers in a multiplex reaction.
The next important feature of desirable primers was to create PCR products of
distinguishable size from the existing primer set for stx1 (210 bp) and stx2 (484 bp).
Based on the size of the target gene, the desirable product size for each additional
pathogen were determined. For L. monocytogenes, the hlyA gene served as a medium
sized target. Therefore a desirable product size for this primer set was between 600 to
23
700 base pairs. The mapA gene of C. jejuni was the smallest gene target and therefore
was required to fall midway between 200 and 400 base pairs to be distinguishable from
the stx1 and stx2 PCR products. The invA gene of Salmonella spp. served as the largest
gene target, so a PCR product greater than 700 base pairs was desirable. The resulting
primers that were designed using our stringent selection criteria are detailed in Table 1.
To ensure that each of these PCR products retain their uniqueness to the organism of
interest the fragments, including the primer sequences, all of the newly selected primers
were analyzed by the BLAST search one last time.
24
Gene/
Primer
Primer Sequence
invA (F)3
invA (R)
4
5’ CCCGATTTTCTCTGGATGG 3’
5’ CATCTTGCTGATGGATTGTTG 3’
16S (F)
5’ CCCCCTGGACGAAGACTGAC 3’
16S (R)
5’ ACCGCTGGCAACAAAGGATA 3’
hlyA (F)
5’ TAGCATCGATTTGCCAGG 3’
hlyA (R)
5’ CTGTAAGTCTCCGAGGTTG 3’
stx2 (F)
5’ GTTTTTCTTCGGTATCCTACCTC 3’
stx2 (R)
5’ CATGCATCTCTGGTCATTGTATTAC 3’
mapA (F)
5’ CACTTTAGACACTGGTATTGCTTTG 3’
mapA (R)
5’ CTTGCTTGGTGCGGATTG 3’
stx1 (F)
5’ TGTAACTGGAAAGGTGGAGTATACA 3’
stx1 (R)
5’ GCTACCTTGAGTCAACGAAAAATAAC 3’
Product
Size1
933
798
641
484
341
210
Tm
°C 2
Reference
60.2
This Study
58.9
This Study
58
52
Wang, 2002
59.2
This Study
61.2
This Study
57.1
59.6
Meng, 1997
59.2
This Study
61.2
This Study
58.9
58.8
Meng, 1997
Table 1. PCR Primers Used in This Study. 1 Product size in base pairs, 2 Tm = melting
temperature, 3 F = forward primer, 4 R = reverse primer.
25
Once the primers were designed, we tested them on DNA templates from both
pure and mixed control cultures. The bacterial strains used as for our control cultures
included: Campylobacter jejuni (Clinical Isolate, University of California Davis),
Salmonella typhimurium (ATTC 14028s), L. monocytogenes (ATTC 396141), and
Enterohemorrhagic E.coli (ATTC 43894). Prior to culturing in liquid media, all strains
except for the microaerophilic C. jejuni, were streak purified on individual Luria Bertani
(LB)(1% Tryptone, 0.5% Yeast Extract, 171.2 mM NaCl) agar plates and incubated
overnight at 37°C. Cultures of C. jejuni were streak purified on Campylobacter selective
CMA agar plates (Remel Incorporated) and incubated overnight at 42°C in
microaerophilic conditions (5% O2 , 5% CO2, 2% H2, and 88% N2) within a
MicroAeroPak (Remel Inc.). Once streak purified, individual isolated colonies of each of
the control strains were transferred to 1 ml of sterile 0.9% saline using a flame-sterilized
loop to create a mixed suspension that would be used as the DNA template for
subsequent PCR analysis.
Multiplex PCR
Multiplex PCR was used to amplify up to five target genes within a single 50 µl
reaction. The initial multiplex PCR design included the identification of STEC, L.
monocytogenes, C. jejuni, and Salmonella spp. Each 50 µl preliminary multiplex PCR
contained 1.0 µl of sample or control milieu culture for the DNA template, 1.0 µl of each
of the foreword and reverse primers for stx1, stx2, hlyA, mapA, and invA (Table 1), 0.5 µl
of 10mM mM dNTP’s (dATP, dCTP, dGTP, dTTP) (Fisher BioReagents exACTGene), 5
26
µl of 10x PCR buffer (5 PRIME; 500 mM KCl, 100 mM Tris-HCl 15 mM Mg(OAc)2,
1% Triton X 100), 34 µl of sterile dH20, and 0.5 µl Taq DNA Polymerase (5Prime; 5
units/µl). For the multiplex PCR reaction that was used to identify STEC and Salmonella
spp only, each 50 µl multiplex PCR reaction contained of 1.0 µl of overnight enriched
swab culture or control milieu culture for the DNA template, 1.0 µl of each of the
foreword and reverse primers for stx1, stx2, and invA (Table 1), 0.5 µl of 10mM dNTP’s
(Fisher BioReagents exACTGene), 5 µl of 10x PCR buffer (5Prime), 38 µl of sterile
dH20, and 0.5 µl Taq DNA Polymerase (5Prime; 5 units/µl). Approximately 30 µl of
sterile mineral oil was used to overlay all of the PCR reactions to prevent evaporation.
The reaction temperature cycles for all multiplex PCR reactions were as follows: 3
minutes at 96°C and 10 minutes at 80°C for bacterial lysis, followed by 35 cycles of 1
minute at 94°C for DNA denaturation, 1.5 minutes at 59°C for primer annealing, and 1
minute at 72°C for polymerase extension. After 35 cycles, the reaction was incubated at
72°C for 10 minutes to allow for final extension (2, 36). Following PCR, all subsequent
reactions were stored at 4°C for short-term storage up to seven days and at -20°C for
longer-term storage until post-PCR agarose gel electrophoresis was done.
All PCR experiments contained both a positive and negative PCR control. For the
positive PCR control, isolated colonies of applicable control cultures Salmonella
typhimurium (ATTC 14028s) , L. monocytogenes (ATTC 396141) , Enterohemorrhagic
E.coli (ATTC 43894) and/or Campylobacter jejuni (Clinical Isolate, UCDavis) were
transferred to 1 ml of sterile LB broth using a flame-sterilized loop to create a mixed
27
suspension for subsequent PCR analysis as detailed above. For the negative PCR control
samples, 1 µl of sterile LB broth was used as the mock DNA template/sample milieu.
PCR for Escherichia coli spp.
We evaluated the presence of Escherichia coli in the overnight enriched swab
cultures as an indicator of fecal bacterial contamination by both PCR and plating
technique. For the PCR analysis, a single PCR assay was utilized to amplify the highly
conserved 16S ribosomal subunit gene within Escherichia coli spp (E. coli). This
reaction was run separately from the multiplex PCR, because the size of the 16S PCR
product overlaps with that of the invA PCR product designed for Salmonella spp in this
study. For the 16S E. coli PCR analysis, each 50 µl PCR reaction was composed of 1.0
µl of sample or control milieu, 1.0 µl of each of the foreword and reverse primers 16S (F)
and 16S (R), 0.5 µl of dNTP’s (Fisher BioReagents exACTGene), 5 µl of 10x PCR buffer
(5Prime), 40 µl of sterile dH20, and 0.5 µl Taq DNA Polymerase (5Prime; 5 units/µl).
Approximately 30 µl of sterile mineral oil was used to overlay each reaction to prevent
evaporation. The reaction time and temperature cycles used were identical to those used
in the multiplex PCR assay for this study.
All E. coli 16S PCRs contained both a positive and a negative control. For the
positive PCR control, isolated colonies of Enterohemorrhagic E. coli (ATTC 43894) were
transferred to 1 ml of sterile LB broth using a flame-sterilized loop to create a mixed
suspension for subsequent PCR analysis, and 1 ul of this milieu was used as template
28
DNA. For the negative PCR control samples, 1 µl of sterile LB broth was used as the
sample milieu.
Post PCR Analysis
To visualize the results of all PCR reactions, 9 µl of each PCR and Multiplex
PCR product plus 1.0 µl of glycerol gel-loading dye (Fisher BioReagents) was loaded
onto a 2% agarose gel in 1% Tris-borate-EDTA (TBE). Additionally, 10 µl of 100bp ladder
(Fisher BioReagents* exACTGene) was used as a molecular weight marker.
Electrophoresis was carried out at 76 Volts for approximately 60 minutes. After
electrophoresis, the gel was stained with Ethidium Bromide and the DNA was visualized
with UV light. Photographs of the gel were taken using a gel documentation system
(Alpha Innotech).
Assessment of PCR Assay Sensitivity and Method Development for Template DNA
Preparation
Sensitivity assays were conducted as part of the method development phase of
this project. Specifically, I wanted to determine the how many bacteria could be detected
using three different sample preparation methods: direct PCR, PCR following bacterial
concentration, and PCR following non-specific enrichment. The most viable method
with the highest recovery achievable would be selected for the primary study.
A serial dilution was created including the following dilutions: undiluted (neat),
10-4, 10-5, 10-6, and 10-7 of each control pathogen (Figure 1). Sterile 5.1 cm x 5.1 cm
rayon/polyester/cellulose gauze swabs (Johnson & Johnson) were used to collect isolated
29
colonies of Salmonella typhimurium (ATCC 14028), and Enterohemorrhagic Escherichia
coli (ATCC 43894) control cultures. Each gauze swab was placed in 20 ml of sterile LB
broth within a labeled 4”x 5” sterile stomacher bag (Seward) and pulsed on low for 60
seconds to create the Neat. For the 10-4 dilution, 19.98 ml of sterile LB broth was
transferred into a stomacher bag. Additionally, 18 ml of sterile LB broth was transferred
into three additional sterile stomacher bags labeled 10-5, 10-6, and 10-7. Sterile gauze
swabs were then added to each. A 20 µl aliquot of the neat solution was added to the 10-4
bag and stomached on low for 60 seconds. A 2 ml aliquot of the 10-4 sample was
transferred to the 10-5 bag and processed in a similar manner. The remaining dilution
series was carried out using the same volume ratios and procedure. The solutions were
then plated on LB agar plates for incubation overnight at 37°C using 100 µl of each
dilution.
To investigate if direct PCR could be utilized on swab samples without further
preparation, 1 µl of each dilution was used in both the multiplex and single PCR
reactions as detailed in Figure 1.
We also wanted to evaluate whether a bacterial concentration and DNA extraction
step would be necessary to perform PCR directly on the sample without enrichment. To
assess this, 100 µl of each dilution was transferred to a 1.5 ml centrifuge tube and
centrifuged at 12,000 rpm for 5 minutes to pellet the cells. The bacteria pellets were
resuspended in 50 µl of 1% Triton X and placed in a heat block at 100°C for 10 minutes.
The tubes were then placed on ice for 10 minutes to cool then centrifuged 12,000 rpm for
5 minutes to pellet cell debris. After decanting off the supernatant of each dilution, 2 µl
30
was used in the both the PCRs (Modified from Wang, 2005). Finally, to examine
whether an overnight enrichment was necessary for detection of the bacteria within the
sample, 1 ml of each control culture dilution was transferred to a corresponding test tube
containing 8 ml of LB broth. The tubes were incubated overnight at 37 °C in a shaking
water bath. The following day, 1 µl of each enriched dilution sample was used in the
PCR reactions (Figure 1).
31
Figure 1. Sensitivity and Method Development Assay. Assay was completed for both
Salmonella typhimurium (ATCC 14028), and Enterohemorrhagic Escherichia coli
(ATCC 43894) control cultures.
32
Experimental Design for Grocery Cart Sampling
A total of twelve grocery stores were selected for this study. The stores selected
represented four grocery story chains each with a store located within three regional
locations east of Sacramento (Figure 2). Each regional location encompassed one or two
smaller cities. The demographics for each region were obtained using the United States
Census 2000 database (Table 2). Two of the grocery store chains represented
discount/outlet grocery establishments and two represented traditional grocery
establishments. The differing store establishments were characterized by the store’s
popularity, shopping environment, as well as self-advertising of the store as belonging to
a specific store type. For example, traditional grocery store establishments maintain high
aesthetic quality in the shopping environment such as accent lighting, decorative flooring,
and attractive product displays. Discount/outlet grocery establishments provide standard
fluorescent lighting, plain floors, and often place pallets of products directly on the sales
floor. Each store was evaluated for the number and organization of carts as well as the
placement of sanitizing wipes and alcohol gel at the entrance to the store and in the raw
meat departments respectively.
33
Figure 2. Map of Regional Locations Selected for Grocery Shopping Cart Sampling.
Region X
Population
Median Household
Region Y
Income
Population
Median Household
Region Z
Income
Population
Median Household
70,673
$73,175
179,250
$61,052
116,115
$48,615
Table 2. Regional LocationIncome
Demographics. Data obtained from 2000 U.S. Census
database (http://www.census.gov/main/www/cen2000.html)
34
Each of the 12 stores was visited five times between March and October of 2009
at approximately two-week intervals (Table 3). The samples were purposefully obtained
during a period of time in which little to no rain events occur to remove rain as a variable
in this study. Ten shopping cart handles were sampled at each store visit for a total of 50
samples collected at each of the twelve stores. In total, 600 samples were collected for
the study.
X
Y
Z
TG
TG
DO
DO
TG
TG
DO
DO
TG
TG
DO
DO
F
G
H
E
J
K
L
A
B
C 10
D 10
I
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Total Carts Sampled
10/20
10/06
09/22
10
10
10
10
10
10
10
10
10
10
10
10
October
September
09/08
10
10
10
10
10
10
10
10
10
10
10
10
08/26
August
10
10
10
10
08/12
07/29
07/08
06/24
July
June
06/10
05/27
May
10
10
10
10
05/13
04/29
04/15
04/01
April
March
1
2
3
4
1
2
3
4
1
2
3
4
03/17
Chain I.D.2
Store I.D.3
Establishment Type1
Region
35
10
10
10
10
50
50
50
50
50
50
50
50
50
50
50
50
Table 3. Store Information, Number of Carts Sampled and Sampling Schedule.
1
Establishment type: DO = discount/outlet type grocery store, TG = traditional grocery
store. 2 Chain ID - Numbers assigned to each of the four grocery store chains sampled.
3
Store ID - Letters assigned to each of the twelve stores sampled.
36
Cart Sampling Method
In an attempt to sample recently used cart handles, grocery shopping carts were
selected for sampling from within customer cart return corrals or from those that were
loose within the parking lot. Cart handles were swabbed using aseptic technique with
moistened sterile gauze (Figure 3). Prior to swabbing, a sterile 4”x 5” stomacher bag
(Seward) was labeled with the store I.D, time, and date of sample. At the time of
sampling, a fresh pair of sterile gloves was used and an individually wrapped, 5.1 cm x
5.1 cm sterile rayon/polyester/cellulose gauze swab (Johnson & Johnson) was removed
from its packaging carefully, avoiding contact with any surface besides the sterile
fingertips of the gloves. Then, 5 ml of buffered peptone water (BPW)(1% Peptone,
85.6mM Sodium Chloride, 24.7mM Disodium Phosphate, 16.5mM Monopotassium
Phosphate, 1000 ml dH20) was applied to the gauze swab from a prepared tube.
The moistened gauze swab was then wiped along the surface of the handle, from
end-to-end, in ten firm horizontal passes (five covering the top half of the handle and five
covering the bottom half – flipping the swab between the top and bottom passes). Great
care was taken to collect surface samples from the sides of handle as well as any
indentation or raised lettering that may be present by pressing the gauze on or into those
surfaces with the fingertips. The swab was then placed into the corresponding labeled
stomacher bag avoiding contact with any other surface including the outside of the bag.
The bag opening was folded over twice and clipped with an extra small metal binder clip
and placed upright within the transport container. The samples were transported to the
lab at ambient temperature and processed within three hours of collection.
37
Method positive and negative control samples were performed at a rate of one per
every five sampling events. For the method positive and negative control, a gauze swab
was prepared for sampling using the exact technique as for cart samples. For the positive
control sample, the moistened gauze was used to collect isolated colonies of Salmonella
typhimurium (ATCC 14028), and Enterohemorrhagic Escherichia coli (ATCC 43894)
from control culture plates within the lab. For the negative method control samples, the
moistened gauze was simply removed from the bag in the lab and returned without
touching any other surface. Both control sample types were subsequently analyzed for
total aerobic bacterial counts and both PCR analyses.
38
Figure 3. Sample Collection Procedure.
39
Assessment of Total Aerobic Bacterial Counts
Each sample was assessed for total aerobic counts (Figure 4). To each sample
bag, 25 ml of sterile Luria Bertani (LB) broth (1% Tryptone, 0.5% Yeast Extract, 171.2
mM NaCl, 500ml dH20) was aseptically added. The bag was then placed in a Seward
Stomacher® 80 BioMaster Lab System (Brinkman, Canada) for 30 seconds on normal
speed.
For each sample collected, two Petrifilm™ aerobic count plates (3M
Microbiology Products) were labeled with the store ID, date collected and dilution
concentration: neat and 1:100. For the neat dilution plating, 1 ml was removed from each
sample bag and applied directly to the labeled Petrifilm™ following the manufacturer’s
instructions. For the 1:100 dilution, 100 μl was removed from each bag and added to 10
ml of sterile BPW. After mixing well by gentle vortexing, a 1 ml aliquot was then
removed from the 1:100 dilution and applied to the Petrifilm™ following the
manufacturer’s instructions. For each batch of Petrifilms™, two negative controls were
performed; one for the neat and one for the 1:100 dilution. A 1 ml aliquot of LB broth
was applied directly to the labeled Petrifilm™ following the manufacturer’s instructions.
For the 1:100 negative control, 100 μl of LB broth was transferred to 10 ml of BPW.
After mixing well, a 1 ml aliquot was then removed from the 1:100 dilution and applied
to the Petrifilm™ following the manufacturer’s instructions. The Petrifilms™ were
allowed to set for 5 minutes, and then were placed in stacks of ten and incubated at 37°C
overnight (18-20 hours).
40
After the homogenized gauze milieu was sampled for total aerobic counts. The
bags containing the gauze were folded over twice and closed with an extra small binder
clip. Each bag was then incubated overnight at 37°C within a shaking water bath set to
80 rotations per minute for overnight enrichment in preparation for PCR.
After overnight incubation, the Petrifilms™ were removed from incubator and the
colonies were counted per the manufacturer’s instruction. For films with less than 250
colonies the exact counts were recorded. For films with greater than 250 colonies, the
average number of colonies per 1cm2 was determined and multiplied by 20 to obtain an
estimated value as per the manufacturer’s instructions.
41
Figure 4. Sample Processing Procedure.
42
E.coli Confirmation Plating Study
In order to confirm PCR results for the E.coli analysis all samples collected on
September 22, 2009 were also analyzed using Petrifilm™ E.coli/coliform Count Plates
(3M Microbiology Products). This culturing media contains Violet Red Bile (VRB) and
an indicator for glucuronidase activity which differentiates E.coli from other coliforms.
For each cart sampled, one Petrifilm™ was labeled with the store ID, and date collected.
After homogenization, 1 ml was removed from each sample bag and applied to the
Petrifilm™ following the manufacturer’s instructions. Two negative controls were
performed; one for the neat and one for the 1:100 dilution. A 1 ml aliquot of LB broth
was applied directly to the labeled Petrifilm™ following the manufacturer’s instructions.
For the 1:100 negative control, 100 μl of LB broth was transferred to 10 ml of BPW.
After mixing well, a 1 ml aliquot was then removed from the 1:100 dilution and applied
to the Petrifilm™ following the manufacturer’s instructions. The Petrifilms™ were
allowed to set for 5 minutes and then placed in stacks of ten and incubated at 37°C
overnight. After overnight incubation the Petrifilms™ were removed from incubator and
the colonies were counted as per the manufactures instruction.
Statistical Analysis
In total, 600 samples were collected at twelve stores within three regional
locations. The twelve stores were grouped by regional location and establishment type.
The establishment type included traditional grocery type stores and discount/outlet type
stores. A two-way analysis of variances (ANOVA) was utilized to determine if there was
43
a statistical significance between regional locations, grocery establishment type, and if
there was relationship between the two (PASW Statistics, Version 18 for Mac, 2-way
ANOVA).
44
RESULTS
Multiplex PCR Primer Design
In this study, we wanted to evaluate the relative levels of bacterial contamination
as well as the specific presence of fecal-bacterial and food-borne pathogen contamination
on shopping cart handles. Because of the sensitivity and ease of high throughput analysis
inherent in molecular diagnostic methods, we chose to use multiplex PCR to detect the
presence of food-borne pathogens. The initial design of our Multiplex PCR assay system
focused on detection of the four most common bacterial causes of food-borne diarrheal
disease in the U.S.: Salmonella spp., STEC, Campylobacter jejuni, and Listeria
monocytogenes. As a starting point for our method, we used the primers for the Shiga
toxin genes stx1 and stx2 developed by Meng et al. 2002 for the detection of STEC.
Previous studies within our laboratory have successfully employed these primers in the
detection of STEC from various sources including ground beef, horse and cattle manure,
and equine hide swabs (2, 26, 30, 36). With the stx1 and stx2 primers in hand, we
developed three other primer sets that detected a specific gene sequence unique to each
organism of interest, did not restrict the binding of the other primers to the target
molecule, and amplified fragment sizes that were distinguishable on an agarose gel when
analyzed by agarose gel electrophoresis.
Based on gene sequence comparisons through the NCBI BLAST
(http://blast.ncbi.nlm.nih.gov/blast.cgi) search function, three unique genes were selected
for amplification by PCR sequence-specific primer targeting. For Salmonella spp., the
invA gene was selected. This gene is highly conserved among the Salmonella spp. and is
45
associated with the adherence and invasion of mammalian cells (20). For L.
monocytogenes, the hlyA gene was selected. This gene product is the well studied poreforming cytotoxin, Listeriolysin O, which is required for escape from intracellular
phagocytic vesicles (1, 7, 31). For C. jejuni, the mapA gene was selected. Although the
mapA gene product has not been identified as a virulence factor, it is a novel membraneassociated protein that is unique to C. jejuni (46). Targeting of this gene allowed for
specific identification of C. jejuni without the possibility of cross-reactivity to a very
similar strain Campylobacter coli. (14, 37, 46, 52).
Utilizing the stringent criteria detailed in the Materials and Method section, three
sets of primers were designed to amplify regions of invA, hlyA, and mapA genes in a
multiplex PCR reaction that would also amplify regions of stx1 and stx2. Once designed,
the primer sets were tested in a five-plex PCR assay using a mixed milieu of individual
overnight cultures of each of the pathogens as template DNA. All predicted fragments
were successfully amplified and resolved by agarose gel electrophoresis (Figures 3 and
4). The next step in our protocol design was to evaluate the sensitivity of the new
Multiplex PCR assay system.
During the development of our overall experimental design for this project, we
chose to modify the list of organisms that we would target for identification with this
study and limit it to the detection of Salmonella spp. and STEC. While infection with L.
monocytogenes is rare, it is fatal in up to 45% of susceptible individuals (CDC
http://www.cdc.gov/salmonella/). Because of the potential risk to the individuals
working in the laboratory at the time this study was carried out, we decided to omit the
46
detection of L. monocytogenes from this study. C. jejuni was also omitted from our list.
C. jejuni is a microaerophilic organism requiring a specialized environment of 3-5%
oxygen and 2-10% carbon dioxide for optimal growth, as well as specialized growth
media (37, 52). After exhaustive efforts in establishing the sensitivity of our assay
system for this pathogen, we decided not to pursue detection of it from the grocery cart
handle swabs. Unfortunately, C. jejuni’s complex culturing requirements were
incompatible with the culturing needs of the other pathogens in this study, and the overall
experimental design for DNA template enrichment from the swabs. Although we
removed L. monocytogenes and C. jejuni from the Multiplex PCR assay in this study,
both sets of PCR primers are available for future studies, and are effective in Multiplex
PCR analysis of samples that do not require enrichment.
47
Figure 5. Five-plex PCR Alignment with Complete and Separate Milieu. Ethidium
bromide stained post-PCR agarose gel with products from multiplex PCR using mixed pathogen
cultures of Salmonella spp., STEC, and L. monocytogenes as DNA templates. (Lane 1) 100 bp
ladder. (Lane 2) mixed -milieu containing Salmonella, Listeria, and STEC. (Lane 3) Salmonella
spp.. (Lane 4) L. monocytogenes. (Lane 5) STEC.
Figure 6. Dilutions of Complete Milieu and Five-plex PCR Alignment. Ethidium
bromide stained post-PCR agarose gel with products from multiplex PCR using mixed
pathogen cultures of Salmonella spp., STEC, L. monocytogenes, and C. jejuni as DNA
templates. (Lane 1) 100 bp ladder. (Lane 2) neat. (Lane 3) 10 -2of milieu, too many to
count (TMTC). (Lane 4)10-4of milieu, Salmonella spp.: 4.12 x104 cfu/ml, STEC:
3.61x104 cfu STEC, L. monocytogenes: 1.83 x 104 cfu, C. jejuni: 7.6 x 103 cfu.
48
Figure 7. E.coli 16S PCR Product Alignment. Ethidium bromide stained post-PCR
agarose gel with products from PCR for E.coli using 16S forward and reverse primers.
Lane 1. 100 bp ladder, Lanes 2-4 STEC.
49
Sensitivity Assays and Method Development
After development of the multiplex PCR, the sensitivity of the assay needed to be
established. The goal of this step was to investigate whether the PCR reactions could be
performed directly on the sample within a few hours of collection, or if additional
bacterial enrichment or DNA purification steps were required. Additionally, we also
wanted to determine how sensitive our Multiplex and 16S PCR analysis could be
utilizing the different enrichment and purification steps. Experiments included PCR
sensitivity tests for direct PCR (data not shown), direct PCR with a bacterial
concentration step (data not shown), and non-specific overnight enrichment (Figure 8,
Figure 9).
50
Figure 8. Sensitivity Assay for Multiplex PCR Non-Specific Enrichment Method.
Ethidium bromide stained post-PCR agarose gel with multiplex PCR products from
dilution series of mixed pathogen cultures of Salmonella spp., STEC, and L.
monocytogenes as DNA templates. (Lane 1) 100bp ladder. (Lane 2) undiluted, (Lane 3)
10-2, (Lane 4) 10-4, (Lane 5) 10-5, (Lane 6) 10-6, (Lane 7) 10-7 = 600 cfu Salmonella spp.,
(Lane 8) 10-8 = 200 cfu E.coli.
Figure 9. Sensitivity Assay for E. coli spp. 16S Non-Specific Enrichment Method.
Ethidium bromide stained post-PCR agarose gel with products from 16S PCR using
pathogen culture of Escherchia coli. (Lane 1) 100bp ladder, (Lane 2) undiluted, (Lane 3)
10-2, (Lane 4) 10-4, (Lane 5) 10-5, (Lane 6) 10-6, (Lane 7) 10-7, (Lane 8) 10-8 = 200 cfu
E.coli.
51
The result of this series of experiments indicated that the non-specific enrichment
step was required to detect Salmonella spp. at low initial concentrations (Figure 8, Figure
9). Using this method, Salmonella spp. was detected at as low as 600 colony forming
units (cfu) per 1 ml and STEC was detected at as low as 200 cfu/ml for Shiga toxin genes
1 and 2 and 200 CFU/ml for the 16S ribosomal RNA target. Based on these findings, the
sample collection methodology and transport for the main study were modified to
accommodate the required non-selective enrichment step. This included transferring the
enrichment broth directly into the stomacher bag that contained the gauze swab used for
sampling to enhance the ability of all of the microbes present to be enriched.
Now that the assay system was developed, and the sensitivity of our assay was
established, we did an initial pilot study to evaluate the assay system in the field. On two
dates, preliminary samples were taken from six different grocery stores raging from one
to three samples taken at each store. A total of ten samples were collected and analyzed
twice using the sample collection, non-selective enrichment, and PCR analysis protocols
detailed in the material and methods section. The results of those analyses indicated a
presence of Escherichia coli spp. on all of the shopping carts sampled based on the
positive amplification of the 16S Escherichia coli ribosomal target sequence (Figure 10,
Figure 11). These results indicated that our assay systems would be effective, and so we
moved into the final stage of our study, applying our methods to a large scale survey of
the Sacramento region.
52
Figure 10. Preliminary Sample Multiplex PCR Results. Ethidium bromide stained postPCR agarose gel with products from preliminary samples. (Lane 1) 100 base pair ladder,
(Lane 2) Positive PCR control with mixed control cultures Salmonella spp. and STEC.
(Lanes 3-13) Preliminary cart samples, (Lane 14) Method negative control, (Lane 15)
PCR negative control.
Figure 11. Preliminary Sample 16S PCR Results. Ethidium bromide stained post-PCR
agarose gel with products from preliminary samples. (Lane 1) 100 base pair ladder,
(Lane 2) Positive PCR control with mixed control cultures Salmonella spp. and STEC.
(Lanes 3-13) Preliminary cart samples, (Lane 14) Method negative control, (Lane 15)
PCR negative control.
53
Prevalence of bacterial contamination on shopping cart handles
Between March and October 2009, six hundred shopping cart handles were
sampled at twelve stores within the Sacramento region. These stores represented three
regional locations, two grocery establishment types and four store chains. All of the
shopping carts sampled were located within the parking lot or within return corrals to
capture recent usage. All six hundred samples were analyzed for number of total aerobic
bacteria, presence of E. coli spp. as an indicator of fecal contamination, and the presence
of STEC and Salmonella spp.
The first portion of our study was to determine the relative level of total aerobic
bacterial contamination on the cart handles. The collected samples were plated onto total
aerobic count films both neat and at a 1:100 dilution. All samples tested resulted in
numbers within measurable ranges for the neat. Total counts ranged from zero to
approximately 53,000 colony forming units (CFU) collected per swab and varied highly
even between carts sampled at the same store on the same day (Table 4, Appendix,
Figure 12, Figure 13).
54
Establishment Type1 Regional Location2
I.D.
TG
DO
X
Y
Z
Mean
4746
3175
4640 3917 3323
Std. Dev.
7424
5807
7957 5747 6173
Table 4. Relative Mean CFU Collected Per Cart and Standard Deviations of Compared
Sample Groups. 1 Establishment Type: TG = Traditional Grocery, DO = Discount
Outlet. 2 See Figure 2 for a map that delineates the regional locations.
55
Comparisons were made between stores by regional location and establishmenttype using the two-way ANOVA (PASW Statistics, Version 18 for Mac, 2-way
ANOVA). The data was grouped by establishment type within each region providing a
replicate value of two (two stores per establishment type within each regional location).
The results of this analysis are presented in Table 5. The analysis revealed that there was
no statistical significance between the number of total aerobic counts found on the
grocery store cart handles in regional locations X, Y, or Z (P > 0.05).
The second analysis compared the amount of total aerobic bacteria found on
grocery store cart handles in stores by establishment-type. The ANOVA statistical
analysis revealed no statistical significance between traditional grocery and discount
outlet establishment types (P > 0.05) for bacterial load.
The third analysis compared the relationship between regional location and store
type. This analysis also revealed no statistically significant relationship (P > 0.05).
Using the same statistical analysis, we also tested the data grouped by sampling event for
each establishment type within regional location allowing us a replicate of five as well as
performing a log transformation of the data prior to analysis (Data not shown). Neither
the five replicate grouping nor log transformation of the data revealed a different result
than the two replicate grouping when analyzed by the two-way ANOVA test.
56
Source of Variation
Regional Location
Establishment Type
Sum of Squares
3.48E+06
7.40E+06
df1
2
1
Mean Square
1.74E+06
7.40E+06
F2
0.541
2.301
P3
0.608
0.18
Regional Location *
Establishment Type
5.72E+06
2
2.86E+06
0.889
0.459
Error
1.93E+07
6
3.22E+06
Total
Corrected Total
2.24E+08
3.59E+07
12
11
Table 5. Results of 2-Way ANOVA. 1df, degrees freedom. 2F, F-value. 3P, P-value or
significance.
20000
Colony Forming Units
15000
10000
5000
1
TG
DO
4
2
X
Y
3
Z
0
Establishment Type
Chains
Regional Location
Figure 12. Combined Mean Total Aerobic Bacteria Count, Grouped by Establishment
Type, Chains, and Regional Location. TG = traditional grocery stores; DO =
discount/outlet grocery stores. Chains 1-4 = chains assigned number for anonymity.
Regions X, Y, Z see Figure 2 for map.
57
Colony Forming Units
20000
15000
10000
5000
F
G
B
C
J
K
E
H
I
A
L
D
0
X
Y
Z
Traditional Grocery
X
Y
Z
Discount/Outlet
Figure 13. Mean Total Aerobic Bacteria Counts per Store, Grouped by Establishment
Type and Regional Location. Stores A-L = stores assigned letter for anonymity. Chains
1-4 = chains assigned number for anonymity. Regions X, Y, Z see Figure 2 for map.
58
The second portion of our study was to determine the presence of E. coli spp. as
an indicator of fecal contamination. Out of 600 samples, 582 were found to be positive
for E. coli spp. (97%). Because the PCR results indicated such dramatically high
prevalence of E.coli spp, we chose to confirm and validate our molecular results with
traditional colony isolation technique. Therefore, the samples that were taken on
September 22, 2009 for PCR analysis were also plated onto Petrifilm™ E. coli/Coliform
Count Plates. As detailed in Table 6, the total counts for E. coli and coliforms paired
with the results of the PCR analysis for presence or absence. These results confirm that
E. coli spp. are present on the majority of the handles of the shopping carts sampled, and
validate our molecular assay detection system for this bacterium. Differences were
observed, however, in that some of the samples, resulted in a positive detection by PCR
for 16S E. coli spp where the Petrifilm™ did not. These data, suggest that our PCR assay
has a higher sensitivity than Petrifilm™ plating methods.
The third and final portion of our bacterial analysis was to identify the presence of
Salmonella spp. and STEC on the surface of grocery shopping cart handles using
Multiplex PCR. Out of 600 samples, one cart was positive for stx1 of STEC (0.17%),
and one cart was positive for Salmonella spp. (0.17%) (Appendix A) Interestingly, both
detections occurred at Store H on the fourth visit on two separate carts (Appendix A).
59
P/A
+
+
+
+
+
+
+
+
+
+
CFU
0
0
30
120
0
30
30
60
30
30
CFU
30
0
30
150
0
60
30
60
30
30
E. coli
PCR
CFU
150
0
60
0
30
30
30
0
30
30
Coliforms
CFU
120
0
60
0
30
30
0
0
0
0
E. coli
P/A
+
+
+
+
+
+
+
+
+
+
H
E. coli
PCR
CFU
0
0
30
30
60
30
120
0
30
0
Coliforms
CFU
0
0
0
30
60
30
90
0
30
0
E. coli
P/A3
+
+
+
+
+
G
E. coli
PCR
E. coli
PCR
Coliforms
CFU
330
420
30
30
60
120
0
30
30
30
Coliforms
CFU2
300
360
0
30
60
90
0
30
0
0
F
E. coli
1
2
3
4
5
6
7
8
9
10
E. coli
Cart #
E1
P/A
+
+
+
+
+
+
+
+
+
+
Table 6. Petrifilm™ E. coli, Total Coliform Counts and PCR Detection Results of
Samples Collected 9/22/2009. 1 Stores assigned E, F, G, and H for anonymity. 2 CFU=
colony forming units 3 P/A = presence or absence; “+” = present, “-” = absent.
60
DISCUSSION
The purpose of this study was to evaluate fecal-bacterial contamination on
grocery shopping cart handles by determining the prevalence of contamination on this
fomite in grocery stores within the Sacramento region. Within the last five years, the
sanitary condition of shopping carts has been launched into public awareness. The 2005
study by Reynolds et al, shocked the public and the grocery industry by declaring grocery
shopping cart handles as one of the most highly biologically contaminated surfaces tested
when compared to other well known “dirty” public surfaces (40). The news articles that
followed fueled that fire (18, 34, 47, 48). Interestingly, within that same year, the
Nonprescription Drugs Advisory Committee (NDAC) under the U.S. Food and Drug
Administration (FDA) met to discuss whether topical hand antiseptics should be
recommended for consumer use (19). The appearance of sanitizing wipes at the front
entrances and sanitizing gels within the meat departments seemed to follow suit.
The epidemiological studies by Jones et al and Fullerton et al., in 2006 and 2007,
that implicated riding in grocery shopping as a risk factor for acquiring traditionally foodborne pathogens in susceptible individuals, took the concern over these surfaces to a
whole new level (21, 27). The evidence now seemed to infer that not only are the
surfaces of grocery shopping cart handles “dirty,” they can also be sources of disease. It
is well known that cross-contamination can occur when handling raw food and that
proper hand-washing amongst the general public can be lacking (6, 8, 9, 24, 16, 28, 29,
41). However, the extension of that knowledge to question the sanitary conditions of
grocery shopping cart handles has only recently begun. While the numerous studies
61
providing evidence for food-borne pathogens on food and food-packaging as well as the
presence of fecal-bacteria on people’s hands highlight the potential for grocery shopping
carts to serve as transmission sources, the evidence linking the carts as transmission
vectors is largely absent. Furthermore, there are no published studies, to date, that have
specifically investigated the type and levels of fecal-borne and food-borne bacteria on
grocery shopping cart handles.
This study is the first, to not only investigate the prevalence of total aerobic
bacterial contamination on the surfaces of grocery cart handles, but also to apply PCR in
the identification of generic E. coli as an indicator for fecal contamination as well as the
identification of the specific food-borne pathogens Salmonella spp. and STEC on this
surface. Our study helps to fill the informational gap between the knowledge of
contamination on food, food packaging, and hands, and the epidemiologically-derived
risk factor of coming into contact with grocery cart handles.
In this study, we successfully developed a molecular method to detect the
presence of generic E.coli spp., STEC, and Salmonella from an enriched culture of the
swab of a grocery cart handle. We then applied this molecular method along with
traditional plating technique for total aerobic bacteria to evaluate bacterial contamination
on grocery cart handles. Our survey included 12 stores in three regional locations east of
Sacramento. Between March and October 2009, each store was visited five times, and
ten carts were selected and sampled at each visit. In total, 600 grocery cart handles were
swabbed for bacterial samples, all of which were successfully analyzed for this study.
62
Our original hypothesis was that the study area would reveal prevalent and high
populations (>106 cfu) of total aerobic bacteria. Overall, the average total aerobic
bacteria recovered from shopping cart handles was near 4000 cfu collected per shopping
cart handle (Table 5, Appendix A). This finding partially supported our hypothesis.
While bacteria were recovered from most cart handles, the numbers recovered were
lower than expected. We expected to find total aerobic bacteria in the millions, whereas
the highest level found was just over 50,000.
One key player in the reduced number of bacteria found may have been the
weather in which the bacteria was deposited and collected. The conditions of weather
such as humidity and temperature are thought to have an effect of the survivability of
bacteria (40, 41). No studies have yet compared the survival of total aerobic bacteria or
E. coli on shopping cart handles in various weather. However, studies have shown that
bacteria survive and even grow on damp surfaces (40, 41). Because our samples were
taken during a time frame with little to no rain, this may explain why the numbers of
bacteria recovered in our study were relatively low. This could also have had an
influential effect on the presence of E. coli as a fecal indicator and the low prevalence of
pathogens STEC and Salmonella spp that was identified. If this explanation is correct, it
would suggests that dry and warm weather may have a beneficial effect on reducing the
numbers of bacteria that could be present. It would be highly useful to conduct a similar
study to include the wet season to determine if the relative level of bacterial
contamination changes to confirm this theory.
63
Interestingly, the results of our study did reveal a wide variance between
individual carts sampled at the same store on the same event. This variability suggests
that contamination levels may also be dependant on the previous user/s of the cart, or the
amount a given cart is used. A number of potential factors may influence the relative
contamination levels between cart users: use of available sanitation options, personal
hygiene, children present, type and number of items placed within the cart, and time span
between use of carts. Further studies are needed to identify which factors play an
influential role. Notably, all stores that were tested in this study offered sanitizing wipes
at the entrance of the stores as well as alcohol-based sanitizing gel within the meat
departments. The actual use of these services may be another one of the reasons why we
identified such low numbers on some of the cart handles, and the high cart-to-cart
variability within our study. An investigation of the prevalence of use of alcohol-based
sanitizers and its effect on bacterial populations on this surface would further support and
encourage retailers in providing these services.
The second goal of this study was to compare the two different grocery
establishment types. The survey of grocery store carts was designed to include four
different grocery store chains. Two of the chains sampled represented traditional grocery
establishments and two represented discount/outlet type grocery establishments. The
reason why we compared these groups was to determine if consumer demographics
influenced hygiene and safe-food handling practices. Public perception of the differences
between the two establishment types is that the discount/outlet type establishments would
be “dirtier”. However, our null hypothesis was that there would be no difference
64
between traditional and discount/outlet grocery establishments. As predicted, no
significant difference was detected between study areas using a two-way ANOVA (Table
4). These results indicate that the store type as well as the regional location of the store
had no effect on the total levels of aerobic bacteria present.
Although not statistically significant, the P value of the establishment type
comparison analysis suggested a trend which was also observed when the average mean
of each store was graphed and the establishment type, regional location, and chains were
grouped (Figure 6, Figure7). Grocery Chain 3, which belongs to the discount/outlet
grocery establishment type, revealed consistently lower levels of total aerobic bacteria
recovered. To determine why this store appeared to have less contamination overall, we
referred back to our evaluation of each store in the study design. Two distinctive
differences of Grocery Chain 3 that stood out in contract to the other stores surveyed
were as follows: the number of carts available, and the method for circulation of carts for
use. It was visually estimated that Grocery Chain 3 had four times the number of carts
available compared to the other three chains. Additionally, recently used carts were
collected from the parking lot and placed at the rear of the designated cart storage area.
The other grocery chains represented, had a return location with a single opening. Carts
were collected from the parking lot and returned to the front of the designated cart
storage area. This meant that the carts from Grocery Chain 3, immediately available for
use, had the potential for a large span of time between users; perhaps even a few days.
Carts immediately available for use from the other chains were likely recently used;
perhaps even just a few minutes. Further investigation is needed to determine if these
65
differences play a significant role in reducing overall level of bacterial contamination on
shopping cart handles.
In addition to evaluating the levels of total aerobic bacteria on shopping cart
handles, we also investigated the prevalence of E. coli spp. Overall, 97% of the samples
tested positive for E.coli spp.. It is important to note that our methodology was only able
to assess presence versus absence and did not measure the population of E. coli bacteria
present. As E. coli is such a ubiquitous organism, its mere presence on a surface is not an
immediate threat. One major source for generic E.coli spp. on the surfaces of grocery
shopping cart handles is likely from the consumer’s hands and is more an indicator of
inefficient hand-washing. The CDC states that ineffective hand-washing is the leading
contributor to the spread of many human pathogens, not just ones of enteric origin. Fecal
contamination on hands in high exposure environments has been well studied and
revealed to be as high as 56% for children in child-care facilities (29). In more transient
environments, such as public transit systems, studies have revealed levels of enteric
bacteria around 28% for adults (28). In summary, the presence of E. coli spp. on hands is
fairly common and we likely come into contact with fecal contamination on a daily basis.
The high prevalence of E.coli spp. found in this study counters previous findings
by Reynolds et al in 2005, where only one of 20% of samples collected from shopping
cart handles tested positive for fecal coliforms (40). In the Reynolds study, total and
fecal coliforms were cultured using selective media. The results of this study suggest that
the use of non-selective enrichment and PCR could provide a more sensitive and robust
method for detecting fecal contamination on surfaces. Our confirmation study utilizing
66
E.coli/coliform Petrifilms™ corresponds with this finding; revealing relatively low or
non-detectable levels of E.coli present within the samples prior to enrichment on that day
(Table 4).
One major subset of organisms that this study did not address is viruses. The
survivability and spread of viruses from fomites, like bacteria, has also been studied (41,
51). Since the results of our study suggest that ineffective hand-washing may be
contributing to bacterial contamination on grocery shopping cart handles, these surfaces
may also serve as transmission vectors for viruses. This is especially important to note as
consumers may not suspect this surface in the transmission of many well known viral
diseases such as those that cause the common cold and flu. A study investigating the
presence of these viruses would be beneficial in understanding the role of grocery
shopping cart handles in the transmission of such diseases.
To take our this study one step further in bridging the gap between the knowledge
of contamination on food, food packaging and hands, and the epidemiologically derived
risk factor with coming into contact with grocery cart handles, we applied multiplex PCR
in the identification of the specific food-borne pathogens Salmonella spp. and Shiga
toxin-producing Escherichia coli (STEC) on the surface grocery cart handles.
Out of 600 samples, one sample tested positive for STEC (0.17%) and one for
Salmonella spp. (0.17%). These results suggest that there is a potential for food-borne
contamination of shopping cart handles. Despite the apparently low incidence of
contamination, the risk for acquiring as infection from this surface is still relevant. The
possibility that infection can result from direct contact with transient contaminated
67
surfaces is supported by the evidence showing that exposure to small numbers of
pathogenic organisms can be sufficient to cause infection (4, 23, 32, 33, 51). While some
microbes require high doses and immediate transmission to infect an individual, some are
infectious at very low numbers and may survive for hours to even weeks on some
surfaces (8). Specifically for the pathogens of interest to this study, the infective doses are
thought to be between ten and 50 cells for STEC (23) and 15 to 20 cells for Salmonella
spp. (CDC. www.cdc.gov/salmonella/. June 8th 2010). Our sensitivity studies reveled that
we could detect STEC at as low as 200 cells and Salmonella spp. as low as 600 cells.
This means that we could have missed detections of these pathogens at levels capable of
causing disease and that are results underestimate the potential for this surface to be
contaminated with these pathogens.
It is also important to point out that this study only looked at two possible foodborne pathogens. The successful identification of Salmonella spp. and STEC on the
surface grocery cart handles suggests that this concept of food-borne pathogens being
present on this surface may be extrapolated to other food-borne pathogens. This concept
is already evident through the epidemiological study by Fullerton et al., citing riding in
grocery shopping carts near meat or poultry as a risk factor for Campylobacter infection
in infants. To further our understanding for the potential of carts to serve as transmission
vectors food borne pathogens, a controlled study specifically identifying the presence of
specific pathogens on the surface food and tracing transference of those pathogens from
the food, to consumer’s hands and to the cart would be beneficial. Additionally, the
sampling of other common surfaces within the grocery store and carts would be useful.
68
Overall, this study highlights the need for further investigation of the sanitary
conditions of shopping cart handles within retail grocery establishments. The results aid
in characterizing the contamination present on these surfaces and should encourage and
further support practices by retailers and consumers to minimize the risks of pathogen
transmission.
69
APPENDIX
70
Appendix: Total aerobic bacteria counts and PCR detection of E. coli, STEC and
Salmonella
Visit #3
Visit #2
Visit #1
Chain ID
Est. Type
Sub Areas
Stores
1
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
4
DO
Y
A
CFU
3,210
7,920
11,940
7,140
3,780
5,040
6,930
6,300
4,950
3,150
2,520
21,000
11,520
18,600
11,400
15,600
4,920
7,200
480
1,470
300
210
60
180
690
210
660
990
120
150
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
1
TG
Z
B
CFU
15,360
8,220
4,110
13,950
8,850
9,240
4,530
660
4,620
5,940
300
90
900
300
60
420
690
330
210
1,620
2,550
4,500
990
3,900
2,670
5,100
4,770
3,300
5,940
4,620
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
2
TG
Z
C
CFU
3,600
3,780
7,290
8,010
4,260
23,760
10,260
1,260
660
750
6,450
14,820
8,130
6,300
3,960
1,380
1,230
750
960
3,150
840
3,300
5,940
540
330
360
4,800
2,100
1,080
1,440
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
3
DO
Z
D
CFU
150
360
450
120
510
180
420
30
210
60
6,300
930
11,880
3,630
2,370
810
720
1,680
540
750
150
240
120
300
300
180
180
150
120
330
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
4
DO
X
E
CFU
2,760
8,400
11,400
20,460
12,600
11,220
10,800
5,700
14,400
18,300
750
570
630
2,040
1,710
930
1,380
960
1,920
3,090
60
30
30
270
270
300
60
180
210
300
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
1
TG
X
F
CFU
9,450
14,820
10,710
1,620
510
25,650
20,730
27,330
6,270
23,820
1,980
3,720
11,400
6,600
19,200
6,420
9,600
16,200
33,600
45,000
1,230
630
810
240
300
180
330
150
300
510
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Grocery Store Chain assigned a number for anonymity. 2 Store Type: TG = Traditional Grocery, DO =
Discount Outlet. 3 See Figure 2 for a map of the regions. 4 Individual Stores assigned a letter for anonymity.
5
CFU= colony forming units. 6 P/A = presence or absence; E = E.coli, S = Salmonella, 1 = STEC stx1.
71
Appendix Continued: Total aerobic bacteria counts and PCR detection of E. coli, STEC
and Salmonella.
Visit #5
Visit #4
Chain ID
Est. Type
Sub Areas
Stores
1
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
4
DO
Y
A
CFU
540
3,600
7,680
2,130
1,020
2,670
1,830
1,260
900
510
7,920
5,940
630
1,890
9,900
6,720
9,240
6,000
5,940
1,530
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
1
TG
Z
B
CFU
1,350
420
2,250
1,110
930
1,530
13,860
12,540
16,500
11,880
1,410
5,940
570
840
450
930
1,440
900
810
660
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
2
TG
Z
C
CFU
3,810
420
330
660
420
240
180
60
240
510
420
540
1,710
2,040
1,680
3,900
4,530
9,240
6,720
3,960
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
3
DO
Z
D
CFU
390
210
2,130
180
240
3,360
210
2,670
720
360
3,960
120
0
240
180
0
30
60
180
30
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
4
DO
X
E
CFU
180
180
210
180
210
300
720
570
210
18,480
5,280
4,500
3,300
2,130
1,800
1,020
810
540
960
1,140
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
1
TG
X
F
CFU
270
330
3,300
570
1,290
930
900
420
930
3,960
23,100
29,040
540
960
14,520
4,620
1,440
4,710
26,400
4,800
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Grocery Store Chain assigned a number for anonymity. 2 Store Type: TG = Traditional Grocery, DO =
Discount Outlet. 3 See Figure 2 for a map of the regions. 4 Individual Stores assigned a letter for anonymity.
5
CFU= colony forming units. 6 P/A = presence or absence; E = E.coli, S = Salmonella, 1 = STEC stx1.
72
Appendix Continued: Total aerobic bacteria counts and PCR detection of E. coli, STEC
and Salmonella.
Visit #3
Visit #2
Visit #1
Chain ID
Est. Type
Sub
Areas
Stores
1
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
2
TG
3
DO
4
DO
1
TG
2
TG
3
DO
X
G
CFU
1,830
2,460
1,740
2,340
3,630
7,020
3,360
2,700
1,260
3,780
43,200
1,080
7,230
7,680
25,800
12,600
33,000
1,320
450
1,200
960
1,980
720
5,940
6,900
6,600
4,620
1,230
420
8,580
X
H
CFU
2,400
2,040
3,600
17,010
1,500
14,490
990
5,400
1,170
22,770
5,400
3,600
720
4,800
2,400
780
360
570
2,820
30
30
240
870
0
540
210
390
630
30
150
Z
I
CFU
8,970
6,210
4,140
9,660
4,830
1,860
3,540
3,450
1,710
4,830
5,280
120
240
420
1,320
17,820
360
360
510
630
570
1,560
4,620
1,290
600
570
270
300
930
570
Y
J
CFU
1,260
1,140
4,200
7,590
4,800
33,450
19,320
3,300
2,760
26,910
1,410
6,330
4,320
990
6,720
9,330
2,520
14,610
7,920
5,400
330
180
360
360
270
180
510
150
810
630
Y
K
CFU
1,410
3,450
19,320
5,520
17,250
37,230
24,840
3,480
1,020
1,320
2,850
3,330
4,950
3,810
7,200
12,300
6,300
10,800
3,120
8,610
1,230
1,050
180
630
720
150
240
3,300
270
210
Y
L
CFU
15,180
13,800
8,970
2,940
3,450
6,900
13,800
3,450
1,380
4,140
2,400
1,230
5,490
1,860
4,230
1,290
930
990
450
390
30
300
180
270
360
360
720
270
210
120
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Grocery Store Chain assigned a number for anonymity. 2 Store Type: TG = Traditional Grocery, DO =
Discount Outlet. 3 See Figure 2 for a map of the regions. 4 Individual Stores assigned a letter for anonymity.
5
CFU= colony forming units. 6 P/A = presence or absence; E = E.coli, S = Salmonella, 1 = STEC stx1.
73
Appendix Continued: Total aerobic bacteria counts and PCR detection of E. coli, STEC
and Salmonella.
Visit #5
Visit #4
Chain ID
Est. Type
Sub
Areas
Stores
1
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
2
TG
X
G
CFU
390
600
420
1,470
1,710
960
630
750
540
270
1,740
690
1,170
960
930
720
450
180
600
780
3
DO
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
X
H
CFU
390
900
60
390
300
150
7,920
4,620
210
30
630
510
180
90
330
150
90
120
510
150
4
DO
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E,S
E
E
E,1
E
E
Z
I
CFU
2,760
390
1,290
6,630
4,620
8,580
19,020
52,800
46,860
2,070
630
9,900
690
270
570
60
0
780
750
810
1
TG
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Y
J
CFU
1,080
1,320
2,700
1,860
3,300
2,130
2,250
1,860
2,310
1,200
2,160
1,110
930
900
1,230
540
870
1,170
1,170
1,020
2
TG
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Y
K
CFU
690
510
480
390
810
630
570
1,860
2,970
900
720
1,080
450
690
420
1,560
60
1,740
570
13,200
3
DO
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Y
L
CFU
6,330
810
870
1,110
3,030
6,090
3,450
3,600
3,990
1,620
570
600
420
420
150
930
270
510
240
240
P
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Grocery Store Chain assigned a number for anonymity. 2 Store Type: TG = Traditional Grocery, DO =
Discount Outlet. 3 See Figure 2 for a map of the regions. 4 Individual Stores assigned a letter for anonymity.
5
CFU= colony forming units. 6 P/A = presence or absence; E = E.coli, S = Salmonella, 1 = STEC stx1.
74
LITERATURE CITED
1. Bansal, N.H., F.H McDonell, A Smith, G. Arnold, G.F. Ibrahim. 1996.
Multiplex PCR Assay for the Routine Detection of Listeria in Food. Int. J. Food
Microbiol. 33:293-300.
2. Barruel, P. 2003. Masters thesis. Isolation and characterization of
enterohemorrhagic E. coli from Sacramento retail ground beef. Thesis. California
State University, Sacramento, CA.
3. Beutin, L., D. Geier, H. Steinruck, S. Zimmermann, and F. Scheutz. 1993.
Prevalence and Some Properties of Verotoxin (Shiga-Like Toxin) – Producing
Escherichia coli in Seven Different Species of Healthy Domestic Animals. J.
Clin. Microbiol. 31:2483-2488.
4. Blanco, J.E., M. Blanco, A. Mora, and J. Blanco. 1997. Production of Toxins
(Enterotoxins, Verotoxins, and Necrotoxins) and Colcins by Escherichia coli
Strains Isolated from Septicemic and Healthy Chickens: Relationship with In
Vivo Pathogenicity. J. Clin. Microbiol. 35:2953-2957.
5. Blanco, J.E., M. Blanco, A. Mora, J. Blanco, and E. A. Gonzalez. 2003.
Verotoxin-Producing Escherichia coli (VTEC) in Spain: Prevalence, Serotypes,
and Virulence Genes of O157:H7 and non-O157 VTEC in Ruminants, Raw Beef
Products, and Humans. Exp. Biol. Med. 228:345-351.
6. Bloomfield, S.F., and E. Scott. 1997. Cross Contamination and Infection in the
Domestic Environment and the Role of Chemical Disinfectants. J. Appl.
Microbiol. 83:1-9.
7. Bubert, A., I. Hein, M. Rauch, A. Lehner, B. Yoon, W. Goebel, M. Wagner.
1999. Detection and Differentiation of Listeria Spp. by a Single Reaction Based
on Multiplex PCR. Appl.Environ. Microbiol. 65:4688-4692.
8. Bures, S., J. T. Fishbain, C. F. Uyehara, J. M. Parker, and B. W. Berg. 2000.
Computer Keyboards and Faucet Handles as Reservoirs of Nosocomial Pathogens
in the Intensive Care Unit. Am. J. Infect. Control. 28:465-471.
9. Burgess, F., C. L. Little, G. Allen, K. Williamson, and R. T. Mitchell. 2005.
Prevalence of Campylobacter, Salmonella, Escherichia coli on the External
Packaging of Raw Meat. J. Food Prod. 68:469-475.
75
10. Cheplic, M. “Kids + Shopping Carts = Stomachaches”. Families.com. Accessed
11 November 2010. http://health.families.com/blog/kids-shopping-cartsstomachaches.
11. Cocolin, L., M. Manzano, C. Cantoni, and G. Comi. 2000b. A Mulitplex-PCR
Method to Detect Enterohemorrhagic (EHEC) and Enteropathogenic (EPEC)
Escherichia coli in Artificially Contaminated Foods. Int. J. Hyg. Environ. Health.
203:159-164.
12. Cornick, N.A., S. L. Booher, T. A. Casey, and H. W. Moon. 2000. Persistent
Colonization of Sheep by Escherichia coli O157:H7 and other E.coli Pathotypes.
Appl. Environ. Microbiol. 66:4926-4934.
13. Darwin, K.H., and V. L. Miller. 1999. Molecular Basis of the Interaction of
Salmonella with the Intestinal Mucosa. Clin. Microbiol. Rev. 12:405-428
14. Denis, M., C. Soumet, K. Rivoal, G. Ermel, D. Blivet, G. Salvat, P. Colin.
1999. Development of an M-PCR Assay for Simultaneous Identification of
Campylobacter jejuni and C.coli. Lett. Appl. Microbiol. 29:406-410.
15. Eklund, M., K. Leino, and A. Siitonen. 2002. Clinical Escherichia coli Strains
Carrying stx Genes: stx Variants and stx-Positive Virulence Profiles. J. Clin.
Microbiol. 40:4585-4593.
16. Ekanem, E.E., H. L. Dupont, L. K. Pickering, B. J. Selwyn, and C. M.
Hawkins. 1983. Transmission Dynamics of Enteric Bacteria in Day-Care Centers.
Am. J. Epidemiol. 118:562-572.
17. Evans D. J., D. G. Evans. 1996. Chapter 25: Escherichia coli in Diarrheal
Disease. Medical Microbiology. University of Texas Medical Branch at
Galveston, Galveston, TX.
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mmed&part=A1408
18. Ewing, S. M. “Germy shopping Carts”. WUSA9.com. 3 December 2008.
Accessed 11 November 2010
http://www.wusa9.com/printfullstory.aspx?storyid=79127
19. FDA, Nonprescription Drugs Advisory Committee (NDAC): Briefing
Document: Benefits and Hazards of Consumer Antiseptic Drug Products.
09/22/2005. http://www.fda.gov/ohrms/dockets/ac/05/briefing/20054184B1_01_01-FDA-Executive%20Summary.pdf
76
20. Fratamico, P.M., and T.P. Strobaugh. 1998. Simultaneous Detection of
Salmonella Spp. and Escherichia coli O157:H7 by Multiplex PCR. J. Ind.
Microbiol. Biotech. 21:92-98.
21. Fullerton, K.E., L. A. Ingram, T. F. Jones, B. J. Anderson, P. V. McCarthy,
S. Hurd, B. Shiferaw, D. Vugia, N. Hubert, T. Hayes, S. Wedel, E. Scallan, O.
Henao, and F. Angulo. 2007. Sporadic Campylobacter Infection in Infants. A
Population-Based Surveillance Case-Control Study. Pediatr. Infect. Dis. J. 26:1924.
22. Galan, J.E., and R. Curtiss. 1991. Distribution of the invA, -B, -C, and -D
Genes of Salmonella typhimurium among Other Salmonella Serovars: invA
Mutants of Salmonella typhi Are Deficient for Entry into Mammalian Cells.
Infect. Immun. 59:2901-2908.
23. Gyles, C.L. 2007. Shiga Toxin Producing Escherichia coli: An Overview. J
Anim. Sci. 85:e45-e62.
24. Harrison, W.A., C. J. Griffith, D. Tennant, and A. C. Peters. 2001. Incidence
of Campylobacter and Salmonella Isolated from Retail Chicken and Associated
Packaging in South Wales. Lett. App. Microbiol. 33:450-454.
25. Heuvelink, A.E., J. T. Zwartkruis-Nahuis, F. L. van den Biggelaar, and W. J.
van Leewen.1999 Isolation and Characterization of Verocytotoxin-Producing
Escherichia coli O157 from Slaughter Pigs and Poultry. Int. J. Food Microbiol.
52:67-75.
26. Hopkins, M.L. 2009. Masters thesis. Prevalence of Shiga-toxin producing
Escherichia coli in range-grazing cattle located in northern California. California
State University, Sacramento, CA.
27. Jones, T.F., A. Ingram, K. Fullerton, R. Marcus, B. Anderson, P. V.
McCarthy, D. Vugia, B. Shiferaw, N. Haubert, S. Wedel, and F. J. Angulo.
2006. A Case-Control Study of the Epidemiology of Sporadic Salmonella
Infection in Infants. Pediatrics. 118:2380-2387
28. Judah, G., P. Donachie, E. Cobb, M. Holland, and V. Curtis. 2010. Dirty
hands: bacteria of faecal origin on commuters' hands. Epidemiol. Infect. 138:40914
29. Kyriacou, A., S. Drakopoulou, I. Georgaki, M. Fountoulakis, E. Mitsou, K.
E. Lasaridi, Y. Manios, and T. Manios. 2009. Screening for faecal
contamination in primary schools in Crete, Greece. Child Care Health Dev.
35:159-63
77
30. Larson, D.G. 2009. Masters thesis. Prevalence of Shiga-toxin producing
Escherichia coli in the Sacramento valley equine population. California State
University, Sacramento, CA.
31. Lawrence, L.M., A. Gilmour. 1994. Incidence of Listeria Spp. and Listeria
monocytogenes in a Poultry Processing Environment and in Poultry Products and
Their Rapid Identification by Multiplex PCR. Appl. Environ. Microbiol.
60:4600-4604.
32. Manning, M.L., L. K. Archibald, L. M. Bell, S. N. Banerjee, and W. R.
Jarvis. 2001. Serratia marcesans Transmission in a Pediatric Intensive Care Unit:
a Multifactorial Occurance. Am. J. Infect. Control. 29:115-119.
33. Manzur, A., L. Gavalda, E. Ruiz de Gopegui, D. Mariscal, M. A. Dominguez,
J. L. Perez, F. Segura, and M. Pujol. 2008. Prevalence of methicillin-resistant
Staphylococcus aureus and factors associated with colonization among residents
in community long-term-care facilities in Spain. Clin. Microbiol. Infect. 14:867 72.
34. Marson, A.E. “Grocery Carts are Germs on Wheels”. Narural News.com 9
September 2010. Accessed 11 November 2010.
http://www.naturalnews.com/029691_grocery_carts_germs.html.
35. Meng, J., S. Zhao, M. P. Doyle, S. E. Mitchell, and S. Kresovich. 1997. A
multiplex PCR for identifying Shiga-like toxin-producing Escherichia coli O157
:H7. Lett. Appl. Microbiol. 24:172-176.
36. Oliver, J. 2000. Masters thesis. Enterohemorrhagic Escherichia coli (EHEC)
detected in retail ground beef in the Sacramento area using Polymerase Chain
Reaction (PCR). Thesis. California State University, Sacramento, CA.
37. On, L.W., P.J. Jordan. 2003. Evaluation of 11 PCR Assays for Species-Level
Identification of Campylobacter jejuni and Campylobacter coli. J. Clin.
Microbiol. 41:330-336.
38. Park, Y.S., S. R. Lee, and Y. G. Kim. 2006. Detection of Escherichia coli
O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in
Kimchi by Multiplex Polymerase Chain Reaction. J. Food Prot. 44:92-97.
39. Read, S.C., C. L. Gyles, R. C. Clarke, H. Lior, and S. McEwen. 1990.
Prevalence of Verocytotoxigenic Escherichia coli in Ground Beef, Pork, and
Chicken in Southwestern Ontario. Epidemiol. Infect. 105:11-20.
78
40. Reynolds, K.A., P. M. Watt, S. A. Boone, and C. P. Gerba. 2005. Occurrence
of Bacteria and Biochemical Markers on Public Surfaces. Int. J. Environ. Health
Res. 15:225-234.
41. Rusin, P., S. Maxwell, and C. Gerba. 2002. Comparative Surface-to-Hand and
Fingertip-to-Mouth Transfer Efficiency of Gram-Positive, Gram-Negative
Bacteria and Phage. J. Appl. Microbiol. 93:585-592.
42. Sacki, B., P. Mensah, and E. Collison. 2001. Campylobacter, Salmonella,
Shigella and Escherichia coli in Live and Dressed Poultry from Metropolitan
Accra. Int. J. Food Microbiol. 71:21-28.
43. Sharma, V.K., and S.A. Carslon. 2000. Simultaneous Detection of Salmonella
Strains and Escherichia coli O157:H7 with Fluorogenic PCR and SingleEnrichment-Broth Culture. Appl. Environ. Microbiol. 66:5472-5476.
44. Sims, K. “Millions of Bacteria Found on Metro Shopping Carts”. KOKO.com.
Accessed 11 November 2010. http://www.koco.com/r/3251843/detail.html
45. Soumet, C., G. Ermel, V. Rose, P. Drouin, G. Salvat, and P. Colin. 1999.
Identification by a Multiplex PCR-based Assay of Salmonella Typhimurium and
Salmonella Enteriditis Strains from Environmental Swabs of Poultry Houses.
Lett. Appl. Microbiol. 29:1-6.
46. Stucki, U., J. Frey, J. Nicolet, A. Burnens. 1995. Identification of
Campylobacter jejuni on the Basis of a Species-Specific Gene That Encodes a
Membrane Protein. J. Clin. Microbiol. 33:855-859.
47. Thompson, C. “Shopping Cart Germs: Overblown or something to worry
about?”. Komononews.com. 14 February 2007. Accessed 11 November 2010.
http://www.komonews.com/news/5819736.html
48. Unknown Author. “Shopping Carts More Germy Than Public Restrooms” Good
Morning America , On Call. 8 February 2007. Accessed 11 November 2010.
http://abcnews.go.com/GMA/OnCall/story?id=2859060
49. Wang, G., C. G. Clark, and F. G. Rodgers. 2002. Detection in Escherichia coli
of the Genes Encoding the Major Virulence Factors, the Genes Defining the
O157:H7 Serotype, and Components of the Type 2 Shiga Toxin Family by
Multiplex PCR. Journal of Clin. Microbiol. 40:3613 - 3619
79
50. Wang, H., and M. F. Slavik. 2005. A Multiplex Polymerase Chain Reaction
Assay for Rapid Detection of Escherichia coli O157:H7, Listeria monocytogenes,
Salmonella typhimurium and Campylobacter jejuni in Artificially Contaminated
Food Samples. J. Rapid Methods Automat. Microbiol. 13:213-223
51. Ward, R., D. D. Bernstein, J. Knowlton, E. Sherwood, T. Yung, T. Cusack, J.
Rubino, and G. Schiff. 1991. Prevention of Surface-to-Human Transmission of
Rotavirus by Treatment with Disinfectant Spray. J. Clin. Microbiol. 29:19911996.
52. Winters, D.K., A.E. O’Leary, M.F. Slavik. 1998. Polymerase Chain Reaction
for Rapid Detection of Campylobacter jejuni in Artificially Contaminated Foods.
Lett. Appl. Microbiol. 27:163-167.