Detection and Enumeration of sublethally-injured Escherichia coli B-41560
using selective agar overlays
A Thesis submitted to the Graduate School in Partial Fulfillment of the
Requirements for the Degree Master of Science
By Amanda Smith
Thesis Advisor: John L. McKillip, Ph.D.
Master’s Thesis
Department of Biology
Ball State University
Muncie, IN
July 2012
Detection and Enumeration of sublethally-injured Escherichia coli
B-41560 using selective agar overlays
A Thesis submitted to the Graduate School
In Partial Fulfillment of the Requirements for the Degree Master of Science
By Amanda Smith
Committee Approval:
___________________________________
Committee Chairperson
Date
___________________________
__________________________________
Committee Member
Date
___________________________
___________________________________
Committee Member
Date
___________________________
Departmental Approval:
___________________________________
Departmental Chairperson Date
___________________________
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Dean of Graduate School
Date
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Ball State University
Muncie, IN
April 27, 2012
2
Table of Contents
I: Acknowledgements……………….………………………………………………… 5
II: Abstract……………………………………………………………………….………. 6-7
III: Introduction
A. Background……………………………………………………….…… 8-9
B. Virulence factors associated with E. coli…………...……. 9-11
C. Detection Methods…………………………………………….…11-15
D. Sublethal Injury…………………..……………………………….15-21
IV: Materials and Methods
A. Growth Curve……………………………………….………………….. 21
B. Heat Shock.…………….………………………………………………… 22
C. Acid Shock……………………...………….……………………….……. 23
D. Petrifilm and Easygel…….…………………………………….. 23-24
E. Food Systems……………………………………………………… 24-25
V: Results
A. Broth System............................................................................... 27-29
B. Beef System………………..……………………….……………….. 29-31
C. IMF (Infant milk formula) System……..…………………... 31-33
VI: Discussion………………………..………..…………………………………….. 33-39
VII: References……...………………………………………………...……………… 40-42
VIII: List of Figures
A. Fig.1 IMS method for isolation of target bacteria……………12
B. Fig. 2 Schematic for PCR amplification of target gene…..…14
C. Fig. 3 Diagram of selective agar overlay method…………….17
D. Fig. 4 Diagram of thing agar overlay method………………… 18
E. Fig.5 Diagram of 4-Tal plating method…………………………..19
F. Fig. 6 Graph of broth results……………………………………….…29
G. Fig. 7 Graph of beef results…………………………………………....31
H. Fig. 8 Graph of IMF results…………………………………………….33
3
I. Table 1 Raw data of colony count results……..………….25-27
4
Acknowledgments
To my family, my parents Darrell and Mary, my sister Meagan and brothers
Chris and Andy, for your never-ending love, support and encouragement. I love you
all and I would not be where I am today without you.
To my grandparents, Jim and Ann Dillhoff, I will be forever grateful to your
everlasting belief that I can be anything I want to be. Your love and support has
made all the difference, I love you both.
To Dr. John McKillip for your guidance, wealth of knowledge and friendship.
Through your example you have taught me the patience to deal with the
unexpected, to appreciate the joys of success and to savor the reward of teaching
others. Words cannot describe my gratitude for the invaluable experience that you
have provided me with, from the bottom of my heart, I thank you.
To Dr. Jim Mitchell for your continuous investment in making this research
the best that it can be. Your valuable experience on the analysis and presentation of
the results has made all the difference. For your time and knowledge, I thank you.
To Dr. Donald Ruch for your time and willingness to serve on my committee.
Thank you for all your beneficial insight, attention to detail and continuous
questions that allowed me to make this research a success.
5
Abstract
Thesis: Detection and Enumeration of sublethally-injured Escherichia coli B-41560
using selective agar overlays
Student: Amanda Renee Smith
Degree: Master of Science
College: Sciences and Humanities
Date: July 2012
Pages: 42
Quality control procedures during food processing may involve either
lengthy enrichment steps, precluding enumeration of bacteria in contaminated food,
or direct inoculation of food samples onto appropriate selective media for
subsequent enumeration. However, sublethally injured bacteria often fail to grow
on selective media, enabling them to evade detection and intervention measures
and ultimately threaten the health of consumers. This study compares traditional
selective and nonselective agar-based overlays versus two commercial systems
(Petrifilm and Easygel) for recovery of injured Escherichia coli B-41560, originally
an isolate from ground beef. Bacteria were propagated in tryptic soy broth (TSB),
ground beef, or infant milk formula (IMF) to a density of 106-108 CFU/mL, and
stressed for six minutes either in lactic acid (pH of 4.5) or heat-shocked for 3 min. at
60°C. Samples were pour- plated in basal layers of either tryptic soy agar (TSA),
Sorbitol MacConkey (SMAC), or Violet Red Bile (VRB) agar and resuscitated for 4h
6
prior to addition of agar overlays. Other stressed bacteria were plated directly onto
the commercial media Petrifilm and Easygel. Our results indicate that the use of
selective and nonselective agar overlays for sensitive recovery and accurate
enumeration of E. coli B-41560 is dependent on the stress treatment and food
system. These data underscore the need to implement food safety measures that
address sublethally- injured bacteria such as E. coli O157:H7, without the use of
enrichment steps, in order to avoid underestimation of true densities for target
pathogens.
7
Introduction
Background:
Escherichia coli (E. coli) is a Gram negative, rod-shaped bacterium that is a
member of the Enterobacteriaceae family (1). Escherichia coli is a facultative
anaerobe and naturally found in the intestinal tract of humans and other mammals.
Escherichia coli within a water system has the ability to cause diarrhea, nausea and
headaches if ingested but can also be more harmful to infants, the elderly and the
immunocompromised. Therefore, E. coli, can be used as an indicator organism to
determine water quality and safety (2.) According to the Environmental Protection
Agency (EPA), the amount of coliforms that can be present in any water sample are
expressly stated and rigidly enforced. These regulations state that the accepted
number of coliforms within a water sample (100 mL) is zero (2,3). As a result of
these regulations, any detectable coliforms, especially E. coli, found within an
intended potable water sample is cause for immediate precautions and treatment to
ensure the safety of consumers (2). Escherichia coli has been found to be
transmissible to humans through many facets, such as fecal contaminated food
(mainly ground beef), contaminated water (as stated above), direct contact with
animals or through person-to-person contact (4).
8
While most strains of E. coli are not harmful to humans, several are
commonly classified into five categories based on their pathogenic mechanisms:
enterotoxigenic, enteroinvasive, enterohemorrhagic (EHEC), enteroaggregative ,
and enteropathogenic (EPEC) (5). The most highly recognized strain of E. coli that
causes disease in humans is Escherichia coli 0157:H7, first reported in 1982 when
associated with morbidity and mortality after isolated from contaminated
undercooked ground beef. In 1993, an outbreak of E. coli 0157:H7 found in
hamburger, caused hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS)
in 500 cases where 151 individuals were hospitalized and three died (6). Although
E. coli 0157:H7 is the most common disease-causing strain in humans, several other
strains are just as harmful. This notion can be shown in data presented by the USDA
Food Safety Inspection Service that reported between 2000 and 2005; the number
of documented toxicoinfections caused by non-0157 STEC (shiga toxin-producing
Escherichia coli) strains in the United States had increased from 171 to 501 cases
during this period (the most recent time frame for which these data are available)
(7).
Virulence Factors associated with E. coli
Escherichia coli has many virulence factors that allow for it to be successful at
causing disease in humans. Not all strains of E. coli possess these virulence factors.
However, it is important to understand the potential for the pangenome to harbor
most or all of these virulence genes, and so, all common virulence factors will be
discussed.
9
Shiga-like toxins (Stx), or verotoxins, are the most well-studied virulence
factor known in regards to E. coli O157:H7. Both Stx1 and Stx2 are made up of six
subunits, one active subunit (A subunit) and five identical binding subunits (B
subunit) (8,9). Escherichia coli 0157:H7 has the ability to produce both Stx1 and
Stx2 (8,9,10) however, this is not always necessary given that Stx2 alone is more
virulent than Stx1 or both produced simultaneously (9,11). It is also important to
note that the aid of the locus of enterocyte of effacement (LEE) and the p0157
plasmid are needed in order for either Stx to produce a toxicoinfection (9). These
toxins are mainly responsible for causing HC and HUS (12). However, these toxins
are frequently augmented by other virulence factors in order to cause elevated
pathogenicity (13). Due to the fact that E. coli must pass through the acidic
environment of the human stomach, some E. coli possess virulence determinants
allowing for survival at low pH (12). These virulence factors include an acid-induced
oxidative system, an acid-induced arginine-dependent system, and a glutamatedependent system (12). Many Shiga toxin-producing E. coli (STEC) strains are
armed with an attaching and effacing (eae) or intimin, gene. This gene product
allows the E. coli strain to attach to ligands or host epithelial cells (1). While
attaching to the cells, strains of E. coli that possess this eae gene produce ‘AE’ lesions
on both intestinal and epithelial cells, which induce structural changes (1). These
changes include loss of intestinal microvilli, pedestal formation, and also a gathering
of cytoskeletal proteins, all of which are responsible for allowing attachment of the
bacteria to host cells (1).
10
Non-0157 STEC are still able to produce the Shiga-like toxins that are known
for E. coli 0157:H7. Although E. coli 0157:H7 is the most recognized strain of E. coli
within the STEC family, non-0157 STEC strains are just as significant and can even
produce the same symptoms as 0157:H7, HUS and even death (14). Non-0157 STEC
has even been identified in 5-63% of sampled food (15) whereas 0157:H7 is shown
to be more rare. This goes to show that although non-0157 STEC are not as highly
recognized, it is still just as important to be aware of these strains.
Detection Methods
Most pathogenic strains of E. coli are able to ferment sorbitol (4). Therefore,
the most common form of isolation of this bacterium from patients or contaminated
food and water sources is by plating the sample on sorbitol MacConkey agar (SMAC)
(1). Although this is a common practice, it is important to note that differences in
growth can occur with different strains of E. coli, such as with the serotype E. coli
0157:H7. Escherichia coli 0157:H7 is one of the few strains of E. coli unable to
ferment sorbitol (16) and therefore will grow on SMAC, but produce colorless
colonies, as opposed to the pink colonies produced by other strains of E. coli that can
ferment sorbitol. Another distinguishing characteristic that E. coli 0157:H7
possesses is the fact that it is negative for the enzyme needed to hydrolyze 4methylumbelliferyl-beta-D-glucuronic acid(MUG) (17), while most other strains of
E. coli demonstrate MUG positive results. To interpret this test a fluorescent light is
used. Escherichia coli 0157:H7 will not fluoresce while other strains of E. coli will.
11
For culture based methods of injured bacteria, it is common that samples
first be nonselectively enriched in trypticase soy broth (TSB) (18), which allows for
the bacteria, if present, to recover/resuscitate. Once the sample has been incubated
in the enrichment broth for several hrs, it is then plated onto selective/differential
media, such as SMAC. Unfortunately, enrichment methods have the potential to be
time-intensive and also negate the ability to quantify the density of the pathogen in
the original food sample.
Culture based methods are not the only method of detection for E. coli.
Immunomagnetic separation (IMS) is another method used for isolation and/or
detection (19). This method utilizes antibodies for E. coli and uses them to coat
ironcored beads. Once the E. coli passes over the beads (or is suspended with them),
bacteria can be captured via a magnetic separation step to bring the bacteria out of
suspension (Fig. 1). The recovered cells are able to be plated on agar, usually SMAC
(20) for enumeration, or subjected to DNA extraction for downstream DNA-based
detection methods. The only downfall of the IMS procedure is that is has heretofore
only been tested using certain strains of E. coli, such as 0157:H7, and may not be as
productive for less virulent strains.
12
Fig. 1 Breakdown of IMS method for isolation of a target bacteria (21).
Another cell immobilization approach is the use of metal hydroxides to aid in
the division of bacteria from the food source (22). This process works by allowing
for the formation of covalent bonds due to placing the appropriate ligand on the
surface of the metal hydroxide in place of the hydroxyl group. This in turn allows for
the adsorption of bacteria to the metal hydroxide (23). Bacteria may be
concentrated from the liquid slurry by centrifugation and plated for enumeration as
in IMS.
Polymerase chain reaction (PCR) is another technique used to evaluate the
presence of E. coli in food and water sources. This procedure is used mainly for
those strains of E. coli that are disease causing, such as the EHEC strains. PCR is
conducted by using specific oligonucleotides, also known as primers, for the
13
purpose of amplifying certain sections of DNA, such as verotoxin or other virulence
genes (24) (Fig. 2).
Target gene
For each cycle:
Denature
Anneal
Extend
Newly synthesized DNA
strand
Oligonucleotide primer
Cycle
1
Cycle
2
Cycle
3
Fig. 2 Basic schematic for PCR amplification of target gene. Adapted from Lynch and
Brown, 1990 (25).
With PCR, it is possible to use samples directly from the food source for
accurate detection enumeration. However, most studies on PCR-based detection of
food pathogens utilize a nonselective enrichment culture first (26). A study
completed by Fortin et. al. showed that sensitivity of detecting EHEC in raw milk and
apple juice improved as enrichment time increased (27). Uttendaele et. al. reported
a similar outcome, revealing that a 6 – 9 hr enrichment step provided a greater
14
sensitivity in detection of EHEC in ground beef (28). An additional study utilizing
beef showed similar results to the previously discussed study. Venkateswaran et. al.
found that PCR detection of E. coli 0157:H7 in a beef slurry showed a higher
sensitivity when permitted to incubate with an enrichment step of up to18 h
compared to using no enrichment (29). However, since nonselective and selective
enrichment steps dramatically increase cell densities prior to DNA template
purification, it becomes impossible to quantify the original density of pathogenic
bacteria in the contaminated food.
Sublethal Injury
Although bacteria are remarkably adaptable to many different environments,
cell injury may easily occur under a myriad of physical and/or chemical conditions.
Bacteria may become injured when some type of stress is brought upon them, which
may greatly alter their physiology (30). Unlike lethally injured bacteria, which are
completely killed and unable to resuscitate, these injured cells may be unable to
grow enough to form colonies when plated directly onto the harsh environment of a
selective medium. However, they would be able to grow well on nonselective media
(31). Under permissive conditions, the sublethally injured bacteria could certainly
be resuscitated if ingested by susceptible hosts in contaminated food. Accurate
detection of injured cells is important for many reasons, one of the most important
being the ability to protect consumers who may ingest food that could be potentially
harmful. Injured cells have the capability to potentially resuscitate, given a favorable
(i.e. host) environment, and become fully virulent.
15
Sublethal injury to bacterial cells can occur by many different methods of
stress. Harsh environmental conditions of stress may include a rapid change in
temperature (cold- or heat-shock), freeze-drying, irradiation, dehydration, osmotic
shock, change in pH, or contact with chemical antagonists such as organic acids (32).
As mentioned previously, nonselective enrichment steps frequently used for
recovery in injured bacterial pathogens are widely used, although any such
approach precludes one from enumerating the original density of bacteria in the
suspect food sample. Thus, the food industry would greatly benefit from a
nonselective recovery approach, which still affords the ability to accurately
enumerate both unstressed and sublethally injured bacteria. Such an approach
would utilize materials and methods already familiar to bench microbiologists,
without the need for an enrichment step nor the added expense and training
required of a PCR-based detection platform.
In recent years, many methods have been used for detection and recovery of
sublethally injured bacteria. Some of these approaches include agar-based methods
such as the overlay playing method, developed by Ray and Speck (33). This method
allows for the bacterial sample to be pour plated with trypticase soy agar (TSA) for
the bacteria to undergo a brief (2-4h) recovery/repair period. Following this short
incubation period, the plates can be overlaid with an appropriate selective medium.
In the case of E. coli in food safety applications, Violet Red Bile (VRB) agar is used
(34). The plates are then incubated at 37C for 24h to allow for growth and
differentiation of coliforms from noncoliform Gram-negatives (31) (Fig. 3). One
16
study compared the plating of dairy products onto VRB or TSA/VRB for the
detection of coliforms (35). The results showed an average of a 20% increase in
detection of coliforms when samples were plated using the selective agar overlay
method (TSA/VRB) compared to plating on VRB alone (35).
Fig. 3 Basic protocol for enumerating sublethally injured bacteria using the selective
agar overlay method (32).
Another method similar to the overlay approach is the surface overlay
method. Here, the sample is spread-plated on TSA instead of being pour plated.
After the allotted repair time, the selective media is then overlaid on the repaired
bacteria and incubated to allow for growth (32). Thin agar overlay, also known as
TAL, is another method used to repair and grow sublethally injured bacteria (Fig. 4).
TAL works by creating a layer of selective media within a Petri dish, allowing for
solidification, and then overlaid with a nonselective media, such as TSA. The injured
bacteria are inoculated directly onto the TSA, which will allow for resuscitation.
17
Once recovered, the repaired bacteria will then interact with the selective media
that has diffused into the TSA layer from underneath (36). Kang and Siragusa found
when E. coli 0157:H7 was inoculated onto beef trim and heat-injured, a significant
difference (p<0.05) was found in the recovery of the bacteria using the selective
agar underlay method compared to plating on straight selective media (37).
Fig. 4 Diagram of the thin agar overlay procedure (36).
Wu and Fung developed a similar method to TAL known as 4-TAL. This
method is the same as the original TAL method except a four-quadrant Petri dish is
employed to allow for the use of more than one selective medium at a time (thin
agar layer) (Fig. 5).
18
Fig. 5 Diagram illustrating the 4-TAL method. XLD = xylose lysine desoxycholate,
CIN = cefsulodin irgasan novobiocin, MOX = modified Oxford medium, MSA =
MacConkey sorbitol agar. (36).
The final agar method used for detection of sublethally injured bacteria is
known as the membrane method. The bacterial sample is inoculated onto a
membrane that is located on top of a plate containing TSA. Once the bacteria have
been resuscitated the membrane is transferred to a selective media. In the study
done by S. E. Hartselle, the selective medium for E. coli was SMAC (31). In a study
using the membrane method E. coli 0157:H7 was sublethally injured by means of a
low or high pH change and inoculated with fermented meat (38). The results
indicated the recovery of the sublethally injured bacteria was enhanced by 3 log
when the membrane was transferred from a nonselective media (TSA) to a selective
medium (SMAC) (38).
19
Commercial methods are currently available to allow for rapid detection of
coliforms. Two methods utilized in this study are the use of Petrifilms and Easygels.
These two methods are currently used within industry to detect E. coli. These two
commercial methods will be compared to more standard based agar methods.
Injured bacteria, such as E. coli, found in foods, have the potential to repair
damaged membranes and other cellular constituents, and produce toxins prior to or
following ingestion in contaminated food (32). Therefore, detection of these
sublethally injured bacteria, regardless of the method, is an important concern not
only for the scientific community, but also for the consumer. The focus of many
investigators in recent years has centered on the idea of developing novel
approaches that incorporate accurate bacterial detection methods during quality
control testing to ensure the safety and health for their consumers (32).
Quality control procedures during food processing may involve direct
inoculation of food samples onto appropriate selective media for subsequent
enumeration. However, sublethally injured bacteria often fail to grow, enabling
them to evade detection and intervention measures and ultimately threaten the
health of consumers. Since false-negative results are potentially dangerous, the main
objective of this research is to compare traditional selective and nonselective agarbased overlays versus two commercial systems (Petrifilm and Easygel) for recovery
of heat- or acid-injured Escherichia coli B-41560.
The hypotheses of this study are the following: a) Selective agar overlays will
recover heat- or acid-stressed Escherichia coli B-41560 with the same efficiency as
20
nonselective media; and b) selective agar overlays will recover heat- or acidstressed Escherichia coli B-41560 with significantly greater efficiency compared to
direct plating onto selective media.
Materials and Methods
Growth Curve
Escherichia coli (E. coli) B-41560 was obtained from the USDA Agricultural
Research Service (ARS) culture collection (Peoria, IL USA) and is considered a
biosafety level 2 (BSL-2) pathogen. Procedures used in culture and maintenance of
this strain were in accordance with the Ball State University Institutional Biosafety
Committee guidelines. This strain of E. coli was originally isolated from ground beef.
Sterile dilution blanks (99 mL of 0.85% NaCl) were used to create serial dilutions of
E. coli in order to construct the growth curve. E. coli was inoculated into a fresh tube
of tryptic soy broth (TSB) (8 mL) (T20-110, Alpha Bio Sciences, Baltimore, MD) and
grown statically overnight at 37C. A subculture was made the next day into fresh
TSB. Predetermined dilutions of E. coli from fresh TSB subcultures were pour-plated
with tryptic soy agar (TSA) (T0502, TEKnova, Hollister, CA) in triplicate every 30
minutes from 0h to 13h. The plates were left to solidify and incubated, inverted, for
24h at 37C. Following 24h incubation, colony counts were conducted. Data were
entered into Microsoft Office Excel for creation and interpretation of the growth
curve.
21
Heat Shock
Escherichia coli was inoculated into fresh TSB and grown overnight at 37C
as described above. A subculture, by sterile inoculating loop, was made the next day
into fresh TSB. Based upon the growth curve, the bacteria were incubated to a
density of 1x106 CFU/mL. 1.5 mL of E. coli was pipetted into a 2mL centrifuge tube
and heated at 58C for 90s with constant swirling. The heat shock procedure used a
400 mL glass beaker filled with 300 mL of water. The water was heated using a hot
plate until the water reached the desired temperature of 58C. Serial dilutions were
prepared and pour plated, in triplicate, with TSA. The plates were left to solidify and
then incubated at 37C for 4h to allow for resuscitation. Following the 4h recovery
period the TSA basal layer was overlaid with another layer of TSA. The overlays
were left to solidify followed by overnight incubation at 37C. The overlay process
was also completed with a TSA basal layer and a violet red bile agar (VRB) (211695,
Difco, Sparks, MD) along with a TSA basal layer and a sorbitol MacConkey agar
(SMAC) (299769, Difco) overlay. Groupings of selective media only were also used
in the overlay process such as a basal layer of VRB with a VRB overlay in addition to
a basal layer of SMAC with a SMAC overlay. Control (nonstressed bacterial) platings,
using the same media combinations were also completed. Colony counts were
obtained for all media groupings, both heat shocked and control plates. Data were
entered into Minitab in order to conduct a one-way analysis of variance (ANOVA).
22
Acid Shock
Escherichia coli was inoculated into fresh TSB and grown overnight at 37C
as described above. A subculture, using a sterile inoculating loop, was made the next
day into fresh TSB. Based on the previously completed growth curve, the E. coli was
incubated at 37C to reach a density of 1x106 CFU/mL. Bacterial culture (6 mL) was
removed and added to a sterile 99 mL 0.85% NaCl blank and inverted to create an
even mixture. A volume of 0.5 L of 85% Lactic acid (0.5 L of 85% v/v)
(W261106,Sigma, St. Louis, MO) was added to the saline blank and inverted to
achieve the desired stress pH of 4.5. The acid/bacterium mixture remained in the
saline blank for 6 min. while inverting every 2 min. to allow for even distribution
and contact of the acid to the E. coli. Sterile saline blanks (99mL) were used to
dilute the bacteria. Aliquots of the diluted samples were transferred onto sterile
Petri plates for TSA pour plating. The same media combinations used in the heat
shock were used for the acid shock procedure. All colony count data was entered
into Minitab in order to conduct a one-way ANOVA.
Petrifilm and EasyGel
Two nontraditional methods of plating were used for the detection of the
nonstressed, heat stressed and acid stressed E. coli in this study. Petrifilm E.
coli/coliform plates (6404, 3M, St. Paul, MN) and EasyGel Coliscan media (25001,
Micrology Laboratories, Goshen, IN) were used in this study to determine their
capability in detecting sublethally injured E. coli using generally accepted ‘rapid’
23
plating systems. Escherichia coli was subcultured from an overnight TSB culture and
heat-shocked using the same protocol applied to the selective agar overlay heat
shock treatment described earlier. The samples to be acid-shocked followed the
same protocol applied to the selective agar overlay acid shock treatment as well.
Aliquots from the serial dilutions were applied directly to the center of the Petrifilm
followed by carefully rolling down the top of the film so as to not allow for bubble
formation. The film was then evenly pressed with the provided pressing tool to
allow for even distribution of the inoculum. Petrifilms were then incubated for 24h
at 37C. The process for the EasyGel media is the same as for the other media in
regards to the heat and acid shock methods. However, once the serial dilutions are
created the sample was added directly to the EasyGel medium, inverted, and poured
onto specially coated Petri dishes provided with the EasyGel medium. The medium
is left to solidify at ambient temperature, and then inverted, incubated, for 24h at
37C. Controls of nonstressed bacteria were completed for both the Petrifilm and
EasyGel media, with both experimental and control groups done in triplicate. Colony
counts were determined and entered into Minitab to allow for the completion of a
one-way ANOVA.
Food Systems
Along with the use of TSB, two food systems were used in this study. These
two food systems were ground beef and nonfat dry milk (NFDM). Lean ground beef
(80g), obtained from local retail, was combined with 80 mL of sterile saline and
24
autoclaved in an Erlenmeyer flask to create a beef slurry. Escherichia coli, grown
overnight in TSB was subcultured using a sterile inoculating loop to transfer the
bacteria into the beef slurry followed by incubation at 37C until the bacteria
reached a density of 1x106 CFU/mL. As with heat shock within the TSB, 2 mL of the
beef slurry was removed and heat shocked for 90s at 58C with constant swirling.
Following the heat shock, dilutions were created and plating was completed using
the same media combinations and alternate plating methods as the TSB experiment.
Controls with no shock were plated for future comparisons. Media were incubated
for 24h at 37C, with colony counts completed the next day and data entered into
Minitab.
For NFDM, 100 mL of reconstituted NFDM powder was autoclaved and kept
in cold storage until needed. Escherichia coli was grown overnight in TSB then
subcultured by looping into the NFDM to allow for growth to reach 1x106 CFU/mL.
The same procedure used for the beef slurry was used here. Acid shock of the
ground beef and NFDM used the same procedure as described for TSB.
Results
Stress Treatment
BROTH
None
None
None
None
None
None
None
None
Dilution
10^-6
10^-5
10^-4
10^-4
10^-6
10^-6
10^-3
10^-6
Plate Type
TSA/TSA
SMAC/SMAC
SMAC/SMAC
VRB/VRB
TSA/VRB
TSA/SMAC
EG
Petrifilm
25
Average (CFU/mL)
5.9x10^7
9.2x10^6
2.29x10^6
6.0x10^5
2.56x10^8
2.47x10^8
2.69x10^5
6.1x10^7
Heat
Heat
Heat
Heat
Heat
Heat
Heat
10^-6
10^-4
10^-5
10^-5
10^-5
10^-6
10^-5
TSA/TSA
SMAC/SMAC
VRB/VRB
TSA/VRB
TSA/SMAC
EG
Petrifilm
5.3x10^7
5.86x10^5
1.04x10^5
1.19x10^7
1.63x10^7
5.5x10^7
1.8x10^6
Acid
Acid
Acid
Acid
Acid
Acid
Acid
10^-4
10^-3
10^-3
10^-5
10^-5
10^-4
10^-4
TSA/TSA
SMAC/SMAC
VRB/VRB
TSA/VRB
TSA/SMAC
EG
Petrifilm
9.8x10^5
1.78x10^5
9.1x10^4
5.7x10^6
6.66x10^6
7.0x10^5
1.55x10^6
BEEF
None
None
None
None
None
None
None
10^-6
10^-6
10^-6
10^-6
10^-6
10^6
10^-6
TSA/TSA
SMAC/SMAC
VRB/VRB
TSA/VRB
TSA/SMAC
EG
Petrifilm
2.75x10^8
4.63x10^7
3.2x10^7
2.57x10^8
2.74x10^8
1.70x10^8
1.29x10^8
Heat
Heat
Heat
Heat
Heat
Heat
Heat
10^-5
10^-5
10^-5
15^-5
10^-5
10^-5
10^-4
TSA/TSA
SMAC/SMAC
VRB/VRB
TSA/VRB
TSA/SMAC
EG
Petrifilm
1.76x10^7
9.9x10^6
6.1x10^6
9.8x10^6
1.48x10^7
1.65x10^7
2.26x10^6
Acid
Acid
Acid
Acid
Acid
Acid
Acid
10^-5
10^-4
10^-4
10^-5
10^-5
10^-4
10^-4
TSA/TSA
SMAC/SMAC
VRB/VRB
TSA/VRB
TSA/SMAC
EG
Petrifilm
4.2x10^6
5.87x10^5
7.37x10^5
4.9x10^6
6.43x10^6
2.25x10^6
2.24x10^6
IMF
None
None
None
None
None
None
10^-6
10^-4
10^-6
10^-5
10^-6
10^-6
TSA/TSA
SMAC/SMAC
VRB/VRB
VRB/VRB
TSA/SMAC
TSA/VRB
1.61x10^8
1.68x10^6
5.1x10^7
2.20x10^7
1.58x10^8
1.62x10^8
26
None
None
10^-6
10^-6
Petrifilm
EG
1.06x10^8
3.8x10^7
Acid
Acid
Acid
Acid
Acid
Acid
Acid
10^-5
10^-3
10^-4
10^-4
10^-5
10^-4
10^-4
TSA/TSA
SMAC/SMAC
VRB/VRB
TSA/SMAC
TSA/VRB
Petrifilm
EG
3.9x10^6
6.2x10^4
2.14x10^6
2.03x10^6
4.3x10^6
1.17x10^6
1.83x10^6
Heat
Heat
Heat
Heat
Heat
Heat
Heat
10^-6
10^-5
10^-5
10^-7
10^-7
10^-6
10^-5
TSA/TSA
SMAC/SMAC
VRB/VRB
TSA/SMAC
TSA/VRB
Petrifilm
EG
7.96x10^7
7.93x10^6
3.6x10^6
3.53x10^8
3.76x10^8
1.03x10^8
5.6x10^6
Table 1. Data displaying raw values for each system (broth, beef and IMF)
under no stress, heat stress and acid stress.
Broth System:
The average number of recovered CFU/mL were determined for both control
(nonstressed) and sublethally injured (heat- or acid stressed) E. coli B-41560 on
each of the media types discussed in the Materials and Methods section. As shown in
Fig. 6, a significant difference in detection among nonstressed bacteria on the
nonselective media combination, TSA/TSA, and the selective agar overlays
TSA/SMAC and TSA/VRB was noted. Interestingly, this difference revealed itself as
a higher rate of recovery on both selective agar overlay combinations (TSA/VRB and
TSA/SMAC) than on TSA/TSA. Conversely, no significant difference was observed
among nonstressed TSA/TSA and the two commercial methods, Easygel and
Petrifilm, as supported by the p-values obtained from a one-way ANOVA. The pvalues obtained for the nonstressed TSA/TSA platings compared to the heat-
27
stressed TSA/TSA showed no significant difference (p =0.765). However, when
comparing nonstressed TSA/TSA to acid-stressed TSA/TSA a significant difference
was noted, and expected, as this reveals the extent of the acid stress on the culture.
(p= 0.006). These results can also be seen in Fig. 6.
Within the subset of heat-stressed bacteria, Fig. 6 reveals no significant
difference in recovery when comparing the nonselective plating, TSA/TSA, to
TSA/SMAC (p=0.065) and TSA/VRB (p=0.124) selective agar overlays. However,
when comparing the nonselective media to SMAC/SMAC (p=0.022) and VRB/VRB
(p=0.043), a significant difference was detected. The same results from the straight
selective media apply to the comparison of TSA/TSA and the commercial method
Petrifilm with a significant difference found. However a significant difference was
found when comparing TSA/TSA to the other commercial method EasyGel
(p=0.884).
Recovered densities on TSA/TSA for acid-stressed E. coli B-41560
demonstrated a significant difference in detection when compared to TSA/SMAC
and TSA/VRB, although again, this was because the counts on the latter two
selective overlay platings were actually higher than those on TSA/TSA (Fig. 6).
Moreover, recovered densities of bacteria following acid stress on these plates were
all significantly higher than those of the straight selective media (SMAC/SMAC ,
p=0.038 and VRB/VRB, p=0.027). TSA/TSA showed no significant difference in
detection compared the commercial methods Easygel (p =0.361) and Petrifilm
28
(p=0.124).
Fig. 6 Recovered density of sublethally injured and non-stressed
Escherichia coli B-41560, cultured in a broth (TSB) system, with various
media combinations. The nonselective media combination, TSA/TSA,
exhibited a significant difference in recovery ability of nonstressed bacteria
compared to the selective agar overlays, TSA/SMAC (p=0.00) and TSA/VRB
(p=0.00).
Beef System:
The average CFU/mL was detected for nonstressed, heat stressed and acid
stressed E. coli B-41560 for all media types. Results differed from those obtained
from the broth system. Fig. 7 shows that in the beef system, recovered bacterial
densities in the nonselective TSA/TSA media combination were significantly higher
than those of either of the selective agars for heat-treated cultures, SMAC/SMAC
29
(p=0.005) and VRB/VRB (p=0.00). The recovery sensitivities of the commercial
method Petrifilm was lower than recovered densities on TSA/TSA control (p=0.00)
The heat-stress treatment exhibited the nonselective media combination to
be significantly higher in recovered E. coli than all other media types, except when
compared to TSA/SMAC and Easygel. The resulting p-values from the one-way
ANOVA show no significant difference between TSA/TSA and TSA/SMAC (p =0.057)
and TSA/TSA compared to Easygel (p =0.45).
Recovery of acid-stressed E. coli in ground beef produced different results
than those that of the heat-stressed bacteria. No significant difference was found
among acid-stressed bacteria plated on TSA/TSA and those plated on the selective
agar overlays, TSA/SMAC (p=0.063) and TSA/VRB (p=0.148). However, a significant
difference was seen in recovered E. coli found among the nonselective TSA/TSA
media type and the straight selective media SMAC/SMAC (p=0.001) and VRB/VRB
(p=0.001) as well as the two commercial plating methods Petrifilm (p=0.006) and
Easygel (p=0.006).
30
Fig. 7 Recovered densities of sublethally injured and nonstressed
Escherichia coli B-41560, grown and stressed within a food system (beef),
and plated onto various media combinations. The nonselective media
combination TSA/TSA, showed no significant difference in detection
compared to the selective agar overlay TSA/SMAC (p=0.709) but there was a
significant difference with TSA/VRB (p=0.00). A significant difference
between TSA/TSA and the commercial plating methods, Easygel (EG)
(p=0.00) and Petrifilm (p=0.00) was also observed.
IMF (Infant milk formula) System:
Figure 8 shows that recovered densities of nonstressed E. coli on TSA/TSA
compared to the selective agar overlays revealed no significant difference,
TSA/SMAC (p=0.534) and TSA/VRB (p=0.83). However, our raw data shows that
counts on TSA/TSA were higher compared to both commercial detection methods
Easygel (p=0.00) and Petrifilm(p=0.00), showing a significant difference among
31
those platings. Within the nonstressed platings Fig. 8 shows there is a significant
drop in the SMAC/SMAC plating combination for recovered densities compared to
the rest of the plating combinations.
For the heat-stressed bacteria, results indicated a significant difference in the
detection of recovered E. coli when comparisons were among TSA/TSA and the
selective agar overlays TSA/SMAC (p=0.001) and TSA/VRB (p=0.003), but as seen in
earlier trials, this is seen as higher recovered bacterial counts in the selective agar
overlay plating combinations compared to TSA/TSA. Likewise, significantly higher
densities were obtained on TSA/TSA when compared to the straight selective
media. SMAC/SMAC (p=0.001) and VRB/VRB (p=0.00). However, TSA/TSA
compared to the two commercial plating methods showed a significant difference
with Easygel (p=0.00) but not with Petrifilm (p =0.054).
The acid-stressed treatment showed a significant difference in recovered
densities with TSA/TSA among all the other media types except when being
compared to TSA/VRB (p =0.422). However, our raw data show recovery of E. coli
on TSA/TSA at 3.9x106 compared to 2-4x106 for the selective agar overlay platings.
This suggests that biologically these plating methods may not be significantly
different although the p value for TSA/SMAC indicates otherwise.
32
Fig. 8 Recovered densities of sublethally injured and nonstressed
Escherichia coIi B-41560, cultured from a food system (infant milk formula),
with various media combinations. The nonselective media combination,
TSA/TSA, showed no significant difference in detection compared to the
selective agar overlays, TSA/SMAC (p=0.534) and TSA/VRB (p=0.83).
However, a significant difference was observed among TSA/TSA and the
commercial plating methods, Easygel (EG) (p=0.00) and Petrifilm (p=0.00).
Discussion
In order to demonstrate the potential use of the selective agar overlay
approach, first the E. coli was grown in TSB and the culture was subjected to heatand acid-stress in this model system prior to moving to the two artificially
contaminated food environments. Comparison of recovery rates among the various
media combinations from the three growth environments could tell a more
complete story of how practical such an overlay approach may prove to be when
33
one wishes to sensitively enumerate viable pathogenic bacteria that have been
subjected to a variety of stressors during food processing. The recovery efficiencies
for TSB-grown lactic acid-shocked E. coli were consistent with our hypotheses. That
is, both selective agar overlay plating regimes recovered higher densities of bacteria
compared to control TSA/TSA densities. While unusual, this result, stemming from
completely separate experiments, nevertheless indicates that acid-shocked E. coli B41560 responds well to a nonselective recovery period. Acid-stressed bacterial
recoveries, when plated directly onto SMAC, VRB, Easygel, and Petrifilm, were not
significantly lower than selective agar overlays, primarily due to the large variances
obtained for the latter plating sets. These results indicate that for this strain of E.
coli, this type of acid stress in TSB does not necessitate a selective agar overlay
method in order to recover comparable levels of E. coli.
Exposure of Gram-negative bacteria to organic acids primarily affects the cell
by first crossing the cell membrane and allowing for the accumulation of the acid
inside the cell (39,40). The accumulation of the acid will lower the intracellular pH
and can potentially inhibit important metabolic reactions, disrupt the proton motive
force or alter the cell membranes protein profile (40). Permitting the bacterial cells
to undergo a brief resuscitation period on nonselective media can allow the
bacterial cell membrane to repair (30, 32) and other mechanisms to be restored
(30). Once the cell is repaired total pathogenicity is then restored (30).
The recovery efficiencies for TSB-grown heat-shocked E. coli were partially
consistent with our hypotheses. Recovered bacterial densities on selective agar
34
overlays, TSA/SMAC and TSA/VRB, were consistently lower in recovery when
compared to the TSA/TSA plating combination (Fig. 6). A difference in detection
abilities was noted however among the selective agar overlays and the straight
selective media platings for the heat-stressed bacteria (Fig. 6). Since sublethally
injured bacteria need a resuscitation period on nonselective media in order to
repair and proliferate (41), this finding supports our hypothesis that the selective
agar overlays produce a higher recovery rate of sublethally injured bacteria
compared to the straight selective media. Similar results were expected when
comparing the selective agar overlays to the two commercial methods. However,
Easygel showed a higher recovery of heat-stressed bacteria compared to the
selective agar overlays, a finding that will require additional consideration.
The larger variances obtained in recovered bacterial densities for VRB/VRB,
SMAC/SMAC, and the two commercial media suggest that the heat treatment used in
TSB-grown culture stress trials may impart a wider variety of physical damage to
the bacteria than the acid treatment. The possibility exists that the E. coli are in
various states and degrees of physical stress, and that the 4h recovery period in the
selective agar overlay trials simply did not improve the plate-based detection
sensitivities. Heat stressing a cell can cause a loss of cellular materials, damage to
wall components as well as the cell membrane (32). Other injuries to the cell
include damage to the ribosome and ribosomal RNA as well as possible damage to
structural DNA (32).
35
The beef food system was used in this study to mimic a scenario by which
one of the most common vehicles for E. coli carriage would be contaminated, and
under situations where the bacteria may be sublethally injured. Our study included
a step wherein the ground beef was inoculated with a very low initial density of
bacteria, and then grown to late log phase in the food environment before stress
treatment and platings. Unlike most studies in the literature, in which the selected
food system is simply spiked at various densities for immediate plating, our study
permitted growth of the pure culture within the food matrix, thus allowing the
bacteria to form intimate associations with the heterogeneous food components.
This approach represents a scenario closer to what would actually be happening in
contaminated food intended for retail sale and consumption.
Within the beef food system a significant difference was detected for
recoveries on TSA/TSA among nonstressed and heat- and acid- stressed bacteria,
thus signifying that the stress treatments are effective. As seen in Fig. 7 the recovery
sensitivities for each medium type (red bars for heat treatment) are nearly identical
(TSA/SMAC p=0.057, TSA/VRB p=0.008). These results do not support our
hypotheses of the selective agar overlays recovering heat-injured E. coli at a higher
efficiency than that of the straight selective media or the commercial methods. As
stated for the TSB results, one can conclude that with this particular strain of E. coli,
after being subjected to heat stress, a resuscitation period on nonselective media or
the use of the agar overlay method is not necessary to improve bacterial recovery. It
is possible that the ground beef components in the food system are in some way
36
serving as a protectant against the heat, buffering the bacteria, as it were, against
the physical denaturing stresses of this temperature and time regime. The acidstress results in ground beef, however, reveal an observation similar to our
hypotheses (Fig. 7). The selective agar overlays recovered similar, or slightly higher
densities of stressed E. coli than did TSA/TSA platings. All the while direct selective
plating on any of the other media types (including the two commercial alternatives)
were significantly less. Thus, for acid-injury in this food system, the use of the
selective overlay method improves detection sensitivity markedly.
Reconstituted infant milk formula, IMF, represents a second food system for
analysis. Our findings indicate that heat-stressed bacteria had higher recovery rates
on the selective agar overlays compared the nonstressed bacteria on the same
media types (Fig. 8). This is an unexpected result, although, as with the ground beef
results, one may conclude that milk solids within the IMF food system are protecting
the bacteria from becoming overly heat-stressed/denatured, and therefore this
treatment is not reducing the viable cell recoveries of bacteria as much as would be
expected. The heat-stress results also show that our hypothesis was correct with
regards to the selective agar overlays having a higher sensitivity of recovery of the
sublethally injured bacteria compared the straight selective media. On the other
hand, Fig. 8 reveals that while the selective agar overlays are more effective in
recovering sublethally injured bacteria when compared to Easygel, Petrifilm seems
to be able to detect the bacteria with the same sensitivity as the overlays. This latter
observation was consistently observed, and will need to be further explored.
37
Fig. 8 shows the recovery of acid-stressed bacteria was similar amongst all
plating groups except for SMAC/SMAC, which exhibited significantly lower recovery
sensitivities. Acid-injured bacteria in IMF were recovered at significantly lower
densities on selective overlay media combinations, although one might submit that,
biologically, these differences are perhaps not meaningful, as the difference is
3.9x106 in the case of TSA/TSA versus 2.03x106 for TSA/SMAC and 4.3x106 on
TSA/VRB plates. Moreover, these differences are complemented by noticing lower
recovery rates on the directly plated straight selective media types (SMAC/SMAC),
the latter sets of which are consistent with our second hypothesis.
In conclusion, it can be determined that the use of selective agar overlays
with this strain of E. coli depends on both the stress treatment and the food
environment/system in which the bacteria is propagated. This statement can be
supported given that within each food system the results varied when looking at
each stressor, either acid or heat.
It can also be determined that there is some sort of strain-to-strain variation
when it comes to the recovery of sublethally injured E. coli. In a pervious study done
in our lab it was found that the selective agar overlay method recovered higher
counts of acid stressed E. coli 0157:H7 in both a broth and ground beef system.
However, within this study it was found that acid stressed E. coli B-41560 would
produce higher colony counts with the selective agar overlay plating method within
a beef system, but not within a broth system. This data could also suggest that the
strain-to-strain variation in recovery may only occur within in a broth system. In
38
future studies, it will be interesting to compare the effectiveness of the selective
agar overlay method for heat stressed E. coli 0157:H7 in beef and also heat- or acidstressed E. coli 0157:H7 in infant milk formula to determine if these speculations
can be upheld.
39
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