1

CHAPTER I

THE PROBLEM AND ITS SCOPE

Background and Rationale of the Study

Burger patties are considered high risk foods that may contain pathogenic

microorganisms and will support the formation of toxins or the growth of pathogenic

microorganisms and foods that may contain harmful chemicals as stated in the Food and Drugs

Administration Circular Order No. 2014-029. When these microorganisms are ingested, the

individual may suffer from gastrointestinal infections.

A wide range of gastrointestinal diseases are caused by bacterial contamination of food.

Foodborne diseases can arise from either infection or intoxication. In both cases, bacterial toxins

are typically responsible for producing disease signs and symptoms. The distinction lies in where

the toxins are produced. In an infection, the microbial agent is ingested which then colonizes the

gut and produces toxins that damage host cells. In an intoxication, bacteria produce toxins in the

food before ingestion. In either case, the toxins cause damage to the cells lining the

gastrointestinal tract, typically the colon. This leads to the common signs and symptoms of

diarrhea or watery stool and abdominal cramps, or the more severe dysentery. Symptoms of

foodborne diseases also often include nausea and vomiting, which are mechanisms the body uses

to expel the toxic materials (“Bacterial Infections”, n.d.).

Common bacterial isolates from burger patties include Staphylococcus aureus,

Salmonella spp., Escherichia coli, Campylobacter spp. among others (“Bacterial Infections”,

n.d.). This study only focuses on E. coli and Salmonella spp. detection due to time constraints.
 2
The bacterium Escherichia coli was first discovered by the German bacteriologist

Theodore von Escherich. E. coli is a gram-negative bacterium and has different strains. Most

strains of E. coli are non-pathogenic and some are even considered as normal flora in the human

intestinal tract, but there are also pathogenic strains of E. coli that may cause complications and

diseases related to the gastrointestinal tract (CDC, 2015).

E. coli is one of the most important examples of a coliform, a bacterial group of the

Enterobacteriaceae family that lives in the GIT of humans and animals and is an essential

microbiological sanitary indicator in product processing and handling hygiene because of its

potential to cause diarrhea or any other illness outside the GIT (Maktabi, Ahangari, & Pour,

2016).

Enterohemorrhagic (EHEC) Escherichia coli (E. coli) O157:H7 are known for their

reputation as a major foodborne pathogen causing infections in the GIT worldwide. The common

EHEC E. coli survives in well-grown cattle which acts as a reservoir. E. coli O157:H7 is a

distinct EHEC strain as it differs in its severity of infection compared to other strains of

Enterohemorrhagic E. coli. Furthermore, the number of foodborne outbreaks related to

pathogenic E. coli proves that E. coli O157:H7 does more damage than other EHEC strains (Lim,

Yoon, and Hovde, 2010).

E. coli O157:H7 is one of the most important enterohemorrhagic strains of E. coli

(EHEC) affecting human health. It is a leading cause of numerous foodborne illness and infantile

diarrhea (Hessain et al., 2015). Not only does it cause diarrhea, it can also cause other

complications and other diseases. The research paper will solely focus on E. coli O157:H7, a

specific EHEC strain, since other EHEC strains do not cause infections and diseases as severe as

E. coli O157:H7.
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Healthy ruminants are the principal reservoir of EHEC, and their fecal contaminations in

meat or dairy products are frequently associated with human infections due to consumption of

such products (Dziva et al., 2004). One of those meat products is ground beef, which is

manufactured into burger patties. The burger is one of the most popular fast food all over the

world, and its patties carry a considerable amount of beef or pork which are kept frozen after

production before being used in restaurants (Edward, Ikpawho, & Ibekwe, 2013). Doing so

prolongs the shelf life of these patties while minimizing contamination and spoilage.

Even adequately cooked meat products are still positive for the presence of coliforms.

Although these cooked meat have been decontaminated, the contamination still happens

especially after cooking. Some contain E. coli that are caused by unsanitary conditions and

preparations. Also, human contact may sometimes introduce E. coli (Badrie, Joseph, & Chen,

2004).

While Enterohemorrhagic Escherichia coli are highlighted in the title to be potentially

present in this study’s burger patty samples, there might also be other pathogenic bacterial

genera present that can also cause infection in the gastrointestinal tract.

Salmonella spp. are among the most common bacteria responsible for foodborne

gastroenteritis and inflammation of the gastrointestinal tract, which makes it a potential

bioterrorism microorganism (Al-Jobori, Al-Bakri, & Al-Baity, 2015). Every year, Salmonella is

also estimated to cause one million foodborne illnesses in United States. Recently, large

outbreaks ground beef as a significant source of multidrug-resistant Salmonella. Most people

infected with Salmonella develop diarrhea, fever, and abdominal cramps twelve to seventy-two

hours after infection (Laufer, Grass, Holt, Whichard, Griffin, & Gold, 2015).
 4
Lim et al. (2010), stated that the total case numbers of EHEC, such as E. coli O157:H7

infections are lower than those of other enteric pathogens such as Salmonella or Campylobacter

spp., but the diseases caused by E. coli O157:H7 showed much higher hospitalization and fatality

rates, thus rationalizing the need to compare and contrast the properties and impact of this

specific serotype to the other two (2) infamous genera.

Several studies have reported that E. coli O157:H7 was able to survive and even grow in

low temperatures. E. coli was subjected to meat that was stored in high temperatures. It showed

that cold and frozen storage is not effective to reduce E. coli O157:H7 in meat samples.

Although there was a decrease in number, it was not significant enough because there are still

more organisms present in the sample (Edward et al., 2013). Aside from other bacterial genera,

non-O157 EHEC are an emerging problem in other countries where they are more prevalent in

compared to the serogroup O157 (Dziva et al., 2004).

Located in a city filled with numerous food establishments, Silliman University also has

various kiosks and stores of its own, spanning the entire main campus. A variety of food is

available for students to choose, and three (3) of these stores provide burgers which are already

cooked and displayed for students to buy. The study intends to determine the physical and

chemical conditions of the ground meat patties before, during and after food processing in the

kiosks under study and the the bacterial load in these different conditions. The results of this

study help in ensuring the quality of food consumption of students in Silliman University, and to

apply precautionary measures needed if results of the aforementioned bacterial genera are found

to be positive.
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Statement of the Problem

Escherichia coli is used as an indicator of food quality and safety, and its presence in

food, which can be a potential bacterial environment, demonstrates the presence of fecal

contamination (Maktabi et al., 2016).

Within the premises of Silliman University, a number of food establishments, including

but not limited to, mini stores or kiosks, sell burgers containing either ground beef or pork

patties. The researchers conducted a study on whether Enterohemorrhagic E. coli or other

pathogenic bacteria, such as Salmonella spp., were present in any of the sold burgers in these

aforementioned places, regardless of the type of meat they used.

The study is only limited to the detection of potentially pathogenic E. coli strains,

specifically Enterohemorrhagic E. coli, and other potentially pathogenic bacteria aside from E.

coli, such as Salmonella spp. The study was applied only to food establishments that sold beef

and/or pork burger patties within the premises of Silliman University, regardless of where and

when the said food establishments got their supply.

Objectives of the Study

To detect if there is presence of bacteria in beef and pork burger patties sold by a number

of food establishments, including, but not limited to, mini stores and kiosks, within the premises

of Silliman University.

The specific objectives are the following:

1.) To determine whether there is a significant difference of microbial load between cooked

and uncooked ground meat burger patties in the different kiosks.

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2.) To determine whether there is a significant difference in the microbial load between

cooked ground meat patties in the different kiosks.

3.) To determine whether there is a significant difference in the microbial load between

uncooked ground meat patties in the different kiosks.

4.) To detect potentially pathogenic bacteria that can cause infection in the gastrointestinal

tract, such as Enterohemorrhagic Escherichia coli and Salmonella spp., between cooked

and uncooked ground meat patties in the different kiosks.

Significance of the Study

The findings of this study will benefit the students of Silliman University considering that

burgers are a popular snack. The greater demand of burgers justify the need for an effective

research and prove that the burgers are under an acceptable level of bacterial load inside Silliman

University. Additionally, the World Health Organization (WHO, 2017) stated that over a billion

cases of gastrointestinal infections have been reported and correlated to millions of deaths every

year.

Thus, burgers sold in kiosks found within Silliman University are tested for the presence

and load of bacteria. For the researchers, the study helps them uncover critical areas related to

their future medical professions and also assists them to expand their knowledge. Also, findings

of this study may be important in planning health education intervention programs for food

handlers and in reducing morbidity and mortality due to foodborne diseases.

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Scope and Limitations

The study compared the microbial load of ground meat patties taken from three (3)

randomly chosen kiosks out of the five (5) kiosks that sell ground meat patties within Silliman

University but was not able to detect the presence of a specific serotype of Enterohemorrhagic

Escherichia coli (E. coli), which is E. coli O157:H7, and other pathogens such as Salmonella

spp., and, Dumaguete City. The samples were subjected to aseptic culture techniques,

biochemical testing, and colony counting that identified and counted any possible

microorganisms that could be found in the burger patties.

In the collection of the different states (uncooked and cooked) of the ground meat patties,

the temperatures and methods of cooking could have varied between kiosks, and these were not

included as a variable since the study only focused on the detection of bacteria in these food

establishments. In the procedure for testing, modifications of the protocol were done due to lack

of resources. Serotyping for the confirmation of E. coli O157:H7 could not be included in the

procedure due to lack of laboratory equipment that could be used, based on the standard

protocol.

Operational Definition of Terms

Aerobic Plate Count - method used in indicating the level of microorganisms in a product.

Burger patties - these are ground meat patties that could be either beef or pork which are

collected from the different kiosks within Silliman University. Both cooked and

uncooked burger patties are the samples needed in order to differentiate which source and

type of operation contains higher microbial load.

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Detection - the process of discovering the presence of potentially pathogenic bacteria, namely E.

coli and Salmonella in burger patties.

Determination - the process of identifying microbial load in cooked and uncooked burger

patties.

Escherichia coli - one of the pathogenic bacteria to be determined in cooked and uncooked

ground meat patties.

Enterohemorrhagic Escherichia coli - a kind of E. coli that causes disease by producing a toxin

called Shiga toxin. This can be measured through the presence of Escherichia coli

serotype O157:H7, in amounts enough to cause bacterial infection in the gastrointestinal

tract which will be determined through colony counting.

Food establishments - kiosks situated within the premises of Silliman University determined to

sell burger patties which are under study.

IMViC - a series of tests used for the differentiation of coliforms which include Indole

production, Methyl red test, Voges-Proskauer test, and the Citrate utilization test.

Microbial load - quantitative analysis of many microorganisms including bacteria, coliform, and

yeast contaminating an object, organism, or organism compartment expressed in

CFU/mL. This can be measured through the use of microbial testing procedure, namely

Aerobic Plate Count.

Pathogenic bacteria - are strains of bacteria that can cause disease to humans. These can be

determined through various biochemical tests in the laboratory. E. coli O157:H7 and

Salmonella spp. are among the two (2) most common pathogenic bacteria associated

with gastrointestinal diseases and other foodborne infections found in ground meat

patties.
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Significant differences in microbial counts - measurable differences of microorganisms

detected between two samples of burger patties in different types of operation, may it be

cooked or uncooked.

Sorbitol-MacConkey (SMAC) - selective growth medium for the differentiation of E. coli

O157:H7 from typical E. coli.

Xylose Lysine Deoxycholate (XLD) - selective growth medium for the isolation of Salmonella

spp. and Shigella spp.

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CHAPTER II

REVIEW OF RELATED LITERATURE

According to the WHO (2017), more than 1.7 billion cases of dysentery around the world

are reported yearly and correlates an estimated 2.2 million deaths. Due to unsafe water supplies,

poor sanitation and hygiene, and nutritional deficiencies, the impediment of diarrheal disease has

become perilous to most developing countries. Although these countries suffer from

gastroenteritis, some high-earning countries still have a significant burden from this infection. A

recent study by Tam et. al (2005) in the United Kingdom showed that 25% of the population

suffered from Intestinal Infectious Disease (IID) which equates to 17 million cases annually.

Approximately 2% of those visit their general practitioner (GP) for consultations account for an

estimated 1 million consultations in a year. 50 % of IID resulted to absences from school and

work due to their symptoms. A comparative was also stated in the study on the percentage of IID

in the 1990’s and in the current decade which showed lesser reported cases yet greater in

communities. This was due to recurring IID symptoms, resulting in them not being able to visit

their GP and a rising number of unreported cases.

The primary symptoms of bacterial gastroenteritis are excessive secretion of fluids from

the small intestine caused by the luminal toxin action, cytotoxic or inflammatory damage of the

ileal or colonic mucosa which might result to a production of blood or pus, and perforation of the

bacterium into the mucosa to the reticuloendothelial system (Humphries & Linscott, 2015).

Escherichia coli is a Gram-negative, rod-shaped, facultative bacterium first described by

Theodor Escherich in 1885. Although most E. coli strains are normal flora of the gastrointestinal

tract (GIT), some have evolved into pathogenic E. coli by acquiring virulence factors and
 11
becoming associated with human disease (Lim et al., 2010). Escherich named it ‘Bacterium coli’

but it was renamed to its present name in 1919 because of bacterial nomenclature revision

(Laury, Echeverry, & Brashears, 2009).

According to the CDC (2018), E. coli consists of a diverse group of bacteria. Pathogenic

E. coli strains are categorized into pathotypes. Six pathotypes are associated with diarrhea and

collectively are referred to as diarrheagenic E. coli:

● Shiga toxin-producing E. coli (STEC) — STEC may also be referred to as

Verocytotoxin-producing E. coli (VTEC) or Enterohemorrhagic E. coli (EHEC). This

pathotype is the one most commonly heard about in the news in association with

foodborne outbreaks.

● Enterotoxigenic E. coli (ETEC)

● Enteropathogenic E. coli (EPEC)

● Enteroaggregative E. coli (EAEC)

● Enteroinvasive E. coli (EIEC)

● Diffusely adherent E. coli (DAEC)

This research paper focuses only on the pathogenic Enterohemorrhagic E. coli pathotype,

as stated in the scope and limitations, to make sure that the researchers do not go beyond the

allotted time to finish the research and also since the method that is used is attainable.

Enterohemorrhagic (EHEC) Escherichia coli or the Shiga toxin-producing E. coli cause

disease by producing Shiga toxin. The most common serotype of EHEC is E. coli O157:H7.

E. coli O157:H7 is one of E. coli’s serious pathogenic serovars in humans (Maktabi et al.,

2016). It is a serotype of Enterohemorrhagic Escherichia coli (EHEC) which was recognized in

1983 as a cause of human disease. EHEC can produce Shiga toxin, which induces attaching and
 12
effacing lesions on intestinal epithelial cells in which the bacteria adhere enterocytes’ apical

surface, and destroying of microvilli. They are associated with diarrhea and hemorrhagic colitis,

which may be complicated by life-threatening renal and neurological sequelae, including

hemolytic uremic syndrome and thrombocytopenic purpura (Dziva et al., 2004).

Escherichia coli O157:H7 is the most common EHEC serotype contributing to >75,000

human infections and seventeen outbreaks in North America per year. Its most serious

complication is the hemorrhagic uremic syndrome (Manning et al., 2007).

E. coli O157:H7 was identified as an agent that caused gastrointestinal illness in 1982.

Outbreaks in Oregon and Michigan that affected forty-seven individuals were caused by the

consumption of contaminated beef patties. Other outbreaks were soon reported and linked to the

consumption of contaminated beef, other foods, water, and contact with animal reservoirs, as

well as person-to-person transmission (Vogt & Dippold, 2005). Oregon and Michigan weren’t

the only places that had an E. coli O157:H7 outbreak in 1982. Cases of other foodborne

outbreaks caused by the bacteria were found in other journal articles.

Escherichia coli O157:H7 was first recognized as a pathogen in 1982 during an outbreak

investigation of hemorrhagic colitis. In 2002, it became nationally notifiable. E. coli O157

outbreak is linked to undercooked ground patties sold from fast food restaurant chain and its

infection can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia,

thrombocytopenia, and renal injury (Rangel et al., 2005). Another E. coli O157:H7 occurred in

1993 and this time it took place in Washington State.

On January 1993, an outbreak of Escherichia coli O157:H7 infections associated with

eating hamburger patties at a fast-food restaurant chain was reported in Washington State. A

number of E. coli O157:H7 have been linked to undercooked ground beef than to any other
 13
vehicle. Several possible approaches may reduce the risk of illness due to this pathogen in meat

products (Shefer et al., 1996). The similarities between these E. coli O157:H7 outbreaks are

simple and distinct. All of the outbreaks have one common source and cause of the bacterium --

meat, specifically ground meat patties in burgers.

Based on a study by Ercolini et al. in 2011, the association between microbial

development and chemical changes occurring during the storage of meat is recognized as a

potential means of revealing indicators of meat quality or freshness. However, the use of chill

temperatures, packaging, and antimicrobials could influence the succession and metabolic

activities of the “ephemeral spoilage microorganisms (ESO)” that are members of spoilage-

associated microbial populations.

According to Lim et al. (2010), contamination is inevitable even way before the

production of the burger patties from meat. From slaughter, beef products may have become

contaminated; the process of grinding beef may cause pathogens to reach the innards of the meat.

If ground beef is incompletely cooked, the bacteria can survive because the heat would not be

able to penetrate deep into the layers and reach the hidden bacteria within.

“One must realize that meat comes from an animal which was skinned,

eviscerated, cooked, then cut into pieces and lastly, carried from one establishment to

another. These manipulations are made in a non-aseptic milieu. Even in taking the best

hygienic precautions, the presence of numerous microorganisms on the surface of the

meat is unavoidable. Since ground meat is, more often than not, made with trimmings,

and since it offers a wider surface to contamination, one can conclude that the microbial

flora of that product would be rather high” (Surkieweiz et al., 1975).

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The process of grinding operations include mixing of different raw beef trimmings from

multiple sources in order to make patties which may carry high pathogenic loads because of their

handling and exposure to multiple exposed surfaces the entire duration. Once again, pathogens

may be dispersed throughout the ground product and there is opportunity for these to multiply in

the subsequent supply chain (Edward et al., 2013).

A lot of factors affect the microbial growth of potentially pathogenic bacteria in meat.

Therefore, it is rather hard to conduct a study about the microbial load of meat, or the patties in

burgers, unless strict control of the conditions and other factors that affect microbial growth and

development are carried out.

Microbial pathogen control in burger patty production poses several challenges. Handling

and preservation can contribute to pathogen growth and transmission. The grinding operation

itself disperses pathogens present on the trimmings throughout the ground product and there is an

opportunity for those pathogens to multiply in subsequent supply chain (Edward et al., 2013).

Moreover, some of the factors that affect the microbial growth in meat do affect the results

greatly. In order to avoid such events, other researchers had to evaluate certain factors.

To know the risk of ground beef preparation, the researchers evaluated on the handling

practices of ground beef, like beef penetration and cooking policies that could lead to cross-

contamination of ready to eat (RTE) food (Bogard et al., 2013).

Another similarity between the food borne outbreaks related to E. coli O157:H7 is that

undercooked ground meat patties were the source of the infection.

A prediction of about 95% of 2.46 billion hamburgers containing 155,000 tons of

Australian beef exported to the United States in 2012 cause illnesses due to undercooking at
 15
home compared to hamburgers cooked at a proper temperature. With a novel approach, E. coli

risk is estimated by using prevalence and concentration estimates in beef withdrawn from the

export chain following pathogen detection (Kiermeier, Jenson, & Sumner, 2004).

The researchers decided that to make use of the given short amount of time and to have

attainable and practical results, the study will only be about the demonstration of potentially

pathogenic serotypes of EHEC, specifically E. coli O157:H7, and other common pathogenic

bacteria that may be present in cooked ground meat patties.

Clinicians should report cases of hemolytic uremic syndrome to their local health

departments as sentinel events for E. coli O157:H7 infection. Restaurants that serve hamburgers

should heed the Food and Drug Administration (FDA) recommendation that all parts of

hamburgers be cooked to an internal temperature of at least 68°C (155°F), and consumers should

be made aware of the potential hazards of eating undercooked ground beef (Cieslak et al., 1997).

Indeed, the severe diseases that E. coli O157:H7 causes needs to be reported to raise awareness,

and those responsible for the unsafe preparation of food that lead to contamination should follow

the guidelines set by the designated organization for food safety.

Another potentially pathogenic bacteria that causes infections in the GIT that the

researchers will include is the Salmonella spp. As stated in the background and rationale part of

the research paper, Salmonella spp. are among the most common bacteria responsible for

foodborne gastroenteritis, an inflammation of the GIT.

Salmonella can be detected by a four-stage process. First stage is the pre-enrichment in

non-selective medium like lactose broth with the addition of Triton X-100. Second, selective

enrichment is done using either Rappaport-Vassiliadis Soy Broth or Tetrathionate broth. Third,
 16
isolation is performed using XLD, BGA, or Hektoen. Last, serological and biochemical

identification of suspected colonies is done for confirmation (Zadernowska & Chajecka, 2012).

To isolate Salmonella spp., sample will be mixed with double-concentrated lactose broth

and inoculated into tetrathionate broth. After, the culture is streaked into XLT4 agar plates

(Apazhao et al., 2001).

Consumption of raw or unsafe food, cross-contamination, improper food storage, poor

personal hygiene practices, inadequate cooling and reheating of food items, and a prolonged time

lapse between preparing and consuming food items were mentioned as contributing factors to an

outbreak of salmonellosis in humans (Ejo, Garedew, Alebachew, & Worku, 2016). The ubiquity

of Salmonella isolates creates a persistent contamination hazard in all raw foods and also in

animal-origin food products, which are often implicated in sporadic cases and outbreaks of

human salmonellosis. Foodborne transmission is recognized as the major cause of Salmonella

infections, with many food sources and supplies implicated in these infections (Ejo et al., 2016).

Theoretical Framework

Bacterial growth in food is usually the result of several chemical and environmental

factors.

“The most important factors that affect microbial growth in foods can be summarized in

the following categories: (i) factors related to the food itself, the “intrinsic factors,” which

include nutrient content, water activity, pH value, redox potential, and the presence of

antimicrobial substances and mechanical barriers to microbial invasion; (ii) factors related to the

environment in which the food is stored, the “extrinsic factors,” including the temperature of

storage, and the composition of gases and relative humidity in the atmosphere surrounding the
 17
food; (iii) factors related to the microorganisms themselves, the “implicit factors,” including

interactions between the microorganisms contaminating the food and between these

microorganisms and the food, e.g., their abilities to utilize different nutrient sources, tolerate

stresses, and produce promoters or inhibitors of growth of other microorganisms, etc.; (iv)

processing factors, which include treatments such as heating, cooling, and drying that affect the

composition of the food and also affect the types and numbers of microorganisms that remain in

the food after treatment; and (v) interaction between the above-described factors can also affect

the growth of microorganisms in foods in a complicated way; the combined effects may be

“additive or synergistic” (Hamad, 2012).

The uncooked and cooked ground meat patties that the researchers tested for this study

have different storage temperatures and time, so they have different temperatures when being

cooked. These are the variables that the researchers have no control over, thus, they are included

in the limitations of the paper. But because of the difference between the factors that affect

microbial growth in food, it is safe to say that bacteria can still grow in an environment wherein

the amount of heat applied to it isn’t enough to kill it.

Various advances in the study of the control of microbial growth have been applied to

many agriculture, medicine and food sciences. The application of the principles of the control of

microbial growth can affect the bacterial load present in burger patties that may contain

potentially pathogenic strains of Escherichia coli and the other pathogenic bacteria such as

Salmonella spp.

Control of microbial growth means to inhibit or prevent growth of microorganisms. This

control is affected in two basic ways: (1) by killing microorganisms or (2) by inhibiting the

growth of microorganisms. Control of growth usually involves the use of physical or chemical
 18
agents which either kill or prevent the growth of microorganisms (Todar, 2004). Agents that

completely kill microorganisms are termed ‘cidal’ while agents that do not completely kill

microorganisms and only inhibit growth are termed ‘static’. Bactericidal refers to the complete

killing of bacteria while bacteriostatic refers to the inhibition of the growth of bacteria.

In microbiology, sterilization refers to the complete destruction or elimination of all

viable organisms in or on a substance being sterilized. There are no degrees of sterilization: an

object or substance is either sterile or not. Sterilization procedures involve the use of heat,

radiation or chemicals, or physical removal of cells (Todar, 2004). The application of heat is the

most effective and widely used method of sterilization. Reduction of bacterial load in food may

be achieved through heating the food or simply through cooking. For sterilization to occur

though, the right amount of temperature and time applied must be considered.

The bacterial load of E. coli O157:H7 in ground meat burger patties can only be

decreased when the right amount of temperature is applied, otherwise, bacteria will not be killed.

A study revealed that at the internal cooking temperatures specified for rare and medium

rare patties, E. coli O157:H7 numbers did not decline significantly compared to those cooked at

71.1°C. Moreover, most consumers determine the doneness of beef patties by observing the color

and texture of cooked meat. However, color is not a good indicator of doneness because ground

beef is prone to a non-typical color change associated with cooking called premature browning

(PMB) (Nair et al., 2016).

The application of the most effective and widely used method of sterilization which is

heat will not be enough to sterilize burger patties and cause a significant decrease in E. coli

O157:H7. Therefore, the possibility of collecting E. coli O157:H7 might still be possible. The
 19
other pathogenic bacteria mentioned in the research paper are readily killed when heated, but

only if the right amount of temperature is applied.

Food should never be held at temperatures between 40° and 140°F, as most bacteria will

quite happily reproduce in that range. It reproduces very slowly, if at all, below 40° and above

140°F. But the temperatures at which bacteria are killed vary according to the microbe. For

example, Salmonella is killed by heating it to 131°F for one hour, 140°F for a half-hour, or

heating it to 167°F for ten minutes. When it comes to killing microorganisms, both heat level and

time affect the equation (Weeks, 2017). Unless the specified temperature and time are applied in

the process of cooking ground meat burger patties, most pathogenic bacteria that may be present

in the patties are likely able to survive.

Conceptual Framework

The convenience and richness in flavors of burgers have made it popular to students.

Despite their popularity, burgers have been rarely studied for their microbiological quality

(Maktabi et al., 2016). There are several factors that may affect the microbiological quality of the

burgers sold inside Silliman University. Some factors that may affect the microbiological

characteristics of burger patties are the sources from where the ground meat was made and where

it was bought. Food handling may contribute to the microbiological quality of the burgers, thus,

it is expected that the samples would have significant differences in microbial count that may

vary between kiosks. The researchers will then determine which kiosk has higher microbial load.

The presence of E. coli and/or other bacteria in ground meat that are sold in the market, if

said meat is not properly handled, is also a factor that contributes in the spread of bacterial

infection in the GIT.

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The state of ground meat patties can be a factor of how high or low the bacterial load of

the patties is. Both cooked and uncooked burger patties will be subjected to food bacteriology

methods to determine which type of preparation has a higher bacterial load.

Several researches prove that cooked ground meat have a slightly lower bacterial load

than uncooked ground meat due to high temperature that is able to penetrate into the meat,

killing some bacteria. Other bacteria, like Salmonella spp., may also be present in meat products

that are made into burger patties.

Figure 1. Conceptual framework of the study.

Hypotheses of the Study

H01: There is no significant difference in the microbial load between uncooked and

cooked states of the ground meat patties.

Ha1: There is a significant difference in the microbial load between uncooked and cooked

states of the ground meat patties.

H02: There is no significant difference in the microbial load between cooked ground meat

patties in the different kiosks.

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Ha2: There is a significant difference in the microbial load between cooked ground meat

patties in the different kiosks.

H03: There is no significant difference in the microbial load between uncooked ground

meat patties in the different kiosks.

Ha3: There is a significant difference in the microbial load between uncooked ground

meat patties in the different kiosks.

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CHAPTER III

METHODOLOGY

Research Design

This study employed the descriptive comparative design to describe differences between

or among variables without manipulating them. The collection of data for the detection of

Escherichia coli and other pathogenic bacteria, such as Salmonella spp., in ground meat patties,

was done using the cross-sectional approach. The study assumes that the design used is

appropriate for describing obtained data.

The Variables of the Study

The dependent variable is the bacterial/microbial load. The independent variable is the

source of the patty as well as the type of preparation of the patty, as in whether it is cooked or

uncooked. The source of the ground meat patties were the food establishments that sold burgers

with ground meat patties situated within the premises of Silliman University.

Sampling Procedure

The purposive sampling technique was used in selecting the samples to be tested. This

technique utilizes the judgment of the researchers to select a sample they believe can provide the

data needed, which was the proper sources for the specimens: the food establishments within the

premises of Silliman University. A letter of consent was sent to the kiosk owners involved before

the collection of the sample.

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Procedures for Data Collection

Sample Collection

For the detection of the presence of the two different organisms to be studied, six (6) burger

patties, composed more of pork than of beef, were taken from three (3) randomly chosen kiosks

around Silliman University’s campus. The samples collected contained equal amounts of cooked

and uncooked burger patties. Three replications were done for the study using the same batch of

burgers from each kiosk. The burger samples from each kiosk were wrapped in sterile plastics

and transported to the laboratory immediately for testing. This ensured that the same sample was

used all throughout the testing.

Figure 2. Flowchart for the collection of samples.

Procedure in Conducting the Laboratory Experiment

The protocols used for all procedures were taken from the Bacteriological Analytical

Manual (BAM) by the U.S. Food and Drug Administration (FDA), with some modifications. For

the aerobic plate count for determination of microbial load, 225 ml of buffered phosphate water
 24
was added to 25 g of sample (1:10 dilution) and homogenized using a sterile plastic bag, and

each sample was placed in separate sterile containers. Three consecutive dilutions (1:100,

1:1000, 1:10000) have been prepared from each sample. 0.5 ml was taken from each dilution and

inoculated to Nutrient Agar using the spread plate method. The plates were then incubated for 48

hours at 35°C. Once growth was observed, colonies were counted manually since the digital

counter was not yet operational.

Figure 3. Flowchart for the determination of microbial load.

The formula used for the conversion of colonies counted in the 1:10000 dilution to

Colony Forming Units per ml is:

CFU/ml = Number of colonies counted x Conversion Factor x Dilution Factor

Inoculum volume

For the detection of E. coli, the same homogenization process was done, 25 g of sample

was added to 225 ml buffered water. Two consecutive dilutions (1:1000, 1:10000) have been

prepared from each sample. Meanwhile, 0.05 ml aliquots were taken from each dilution and

inoculated onto an Eosin Methylene Blue agar. These plates were then incubated for 18-24 hours
 25
at 37°C. Typical E. coli colonies appear blue-black with a greenish metallic sheen on EMB. Said

typical E. coli colonies that grew after 18-24 hours were picked and subcultured onto a Sorbitol

MacConkey Agar. Typical E. coli colonies are pink on S-MAC, but O157:H7 serotypes appear

colorless, as they do not ferment sorbitol. When colorless colonies were observed after 18-24

hours, the said colonies were picked and inoculated onto EC broth. If E. coli is suspected, the EC

broth will become turbid. Turbidity was not observed after 18-24 hours of incubation; thus the

organism was not subjected to further testing through the use of IMViC tests (Indole, Methyl

Red, Voges-Proskauer, and Citrate).

 26

Figure 4. Flowchart for the detection of E. coli. O157:H7

 27
For the detection of Salmonella, the same homogenization process as that of E. coli was

used, except that the diluent used was sterile Lactose Broth. The lids of the containers were

loosened before incubation, and the samples were incubated for 24 hours at 35°C. Once

incubation was completed, the lids were tightened, and the samples were shaken gently. 0.1 ml

was then taken from the sample and inoculated onto a Xylose Lysine Deoxycholate (XLD)

medium, and another 1 ml was inoculated onto a 10 ml tetrathionate broth medium. The XLD

agar plates and TT broth tubes were then incubated at 35°C for 24 hrs. Characteristic colonies of

Salmonella spp. were not seen after incubation, so further testing was no longer conducted. After

incubation, 10 μl of TT broth was no longer streaked onto a Bismuth Sulfite Agar (BSA) and

Salmonella-Shigella Agar (SSA) nor repeated for the XLD agar.

 28

Figure 5. Flowchart for the detection of Salmonella spp.

 29
Data Analysis

Analysis of the data was done using the Two-way t-test for dependent variables and One-

Way Analysis of Variance (ANOVA) for independent variables.

The Pair-difference t-test was used in the study to compare the difference of the average

means of the dependent variables. The One-way Analysis of Variance (ANOVA) was used in the

study to determine whether there was any significant difference between the means of the

independent groups of the research.

For the data on the presence of E. coli O157:H7 and Salmonella spp., a checklist was

made since the study only detected the presence of these bacteria. The growth of these bacteria

was based on the description of the colonial morphology from the protocol.

Ethical Considerations

Through the duration of the study, the food establishments were guaranteed full

anonymity. Thorough briefing and explanations as to the rationale behind the study were made

aware of the chosen food establishments to give them the freedom of choice whether to

participate in the study or not. Participants were informed that any data provided was kept with

utmost secrecy. Aside from that, the food establishments were reminded that failure to

participate in the study would not result to any fines or consequences and that despite already

consenting, they had the right to withdraw from the study and refuse to provide any information.

Furthermore, the consent and scope of the study were approved by the University Research

Ethics Committee (UREC).

 30
CHAPTER IV

RESULTS AND DISCUSSION

This chapter presents the data gathered, statistical analyses and interpretation of both

quantitative and qualitative analyses.

To determine the significant difference of microbial load between cooked and uncooked

ground meat burger patties in the different kiosks.

Hamad (2012) stated that there are certain factors that affect microbial load in food. First,

factors that are related to the food itself are called “intrinsic factors”, these include nutrient

content, water activity, pH value, redox potential, and the presence of antimicrobial substances

and mechanical barriers to microbial invasion. Second, factors related to the environment where

food is stored and kept, including the temperature of storage, and the composition of gases and

relative humidity in the atmosphere surrounding the food are called the “extrinsic factors”. Third,

factors related to the microorganisms themselves, including interactions between the

microorganisms contaminating the food and between these microorganisms and the food are

called “implicit factors”. Fourth, processing factors, which include treatments such as heating,

cooling, and drying that affect the composition of the food and also affect the types and numbers

of microorganisms that remain in the food after treatment. Lastly, the interaction between the

above-described factors can also affect the growth of microorganisms in foods in a complicated

way; the combined effects may be “additive or synergistic”.

For the detection of microbial load in CFU/ml in cooked and uncooked burger patties,

three (3) consecutive dilutions were made. Appendix _ shows the microbial load in CFU/ml in
 31
all three dilutions for the three replications. The data in Table 1 shows the microbial load in

CFU/ml in cooked and uncooked burger patties in 1:10000 dilution for the three replications.

The researchers decided to utilize the microbial load in CFU/ml in the 1:10000 dilution only

since some of the results were inconclusive in the 1:100 and 1:1000 dilutions (see Appendix D

for the complete results of the raw data of microbial load in CFU/ml in all three dilutions for the

three replications). Possible procedural errors could’ve caused some of the inconclusive results.

Table 1.Microbial load in CFU/ml in cooked and uncooked burger patties in 1:10000 dilution.
Kiosk Uncooked Cooked
S1 2.80 x105 3.02 x106 1.00 x107 0 6.06 x106 3.20 x105
1 S2 4.00x104 1.61 x107 5.34 x106 0 1.63 x107 6.00 x104
S3 9.40 x105 2.74 x106 2.82 x106 0 1.60 x105 2.00 x104
S1 0 3.14 x106 7.00 x106 0 4.64 x106 7.20 x105
2 S2 0 7.40 x105 1.14 x106 0 2.74 x106 6.02 x106
S3 1.50 x106 3.60 x105 7.04 x106 0 2.80 x105 6.00 x104
S1 0 2.08 x106 5.38 x106 0 2.80 x105 5.06 x106
3 S2 8.00 x104 9.60 x106 2.32 x107 1.00 x105 1.06 x106 4.18 x106
S3 2.00 x104 3.40 x105 1.80 x105 0 9.24 x106 3.35 x107

In line with the results of Table 1, the data shown in Table 2 shows that there is no

significant difference in the microbial load between the uncooked and cooked burger patties.

This is indicated in the p value of 0.94, which is greater than the level of significance set at 0.05.

In order for a significant difference to exist, the p value must be lesser than 0.05. Such finding is

further confirmed by the tabular/critical value of 2.12, which is greater than the t statistics, 0.08.

Since the p value is greater than 0.05, the null hypothesis for the difference in microbial

load between cooked and uncooked burger patties is accepted. This means that even after the
 32
application of heat, microorganisms were still present in the cooked burger patties. Most of the

samples also had alarmingly high numbers of microbial load in both the uncooked and cooked

samples,, which means that when an immunocompromised individual happens to have eaten the

contaminated burgers, this may lead to complications or illnesses in the gastrointestinal tract.

De Jong et. al (2012) stated that improper cooking is one of the main factors causing food

borne illness. This is partly caused by the consumption of undercooked meat. Most consumers do

not use a meat thermometer but determine the doneness of meat most often by cutting the meat

to evaluate changes in color and texture, or by other subjective techniques.

The temperature in which the patties are cooked and the length of time it took to cook

them are the factors that most likely contributed to the growth of microbes even after the burger

patties have been heated. Other factors such as the ability of certain bacteria to survive in

extreme temperatures, or whether the equipments used were sterile or not may also be the

possible causes.
 33
Table 2. T-test results of the difference in the microbial load between the uncooked and cooked
burger patties.
Uncooked Cooked
Mean 3494074.07 3363703.70
Variance 7.22E+12 1.92E+13
Observations 9 9

Hypothesized Mean Difference 0

df 16
t Stat 0.08
P(T<=t) two-tail 0.94

t Critical two-tail 2.12

To detect significant difference of microbial load between cooked ground meat patties in

the different kiosks.

The data in Table 3 shows the summary of the results of the microbial load between

cooked burger patties in the different kiosks from the 1:10000 dilution. The microbial load in the

1:10000 dilution for the three replications were added and the average and variance were

computed.

Table 3. Summary of results of the microbial load between cooked burger patties in the different
kiosks (1:10000 dilution).
Groups Count Sum Average Variance

Kiosk 1 3 7633333 2544444 7.38E+12

Kiosk 2 3 4820000 1606667 1.99E+12

Kiosk 3 3 17820000 5940000 5.19E+13

 34
The data in Table 4 shows that no significant difference exists among the three kiosks

which serve as the sources of the cooked burger patties. This is indicated in the p value of 0.507

which is greater than the level of significance at 0.05, which is also confirmed by the critical

value of 5.143 which is greater than the computed or the 0.763 F statistics.

Since the p value is greater than the level of significance, the null hypothesis for the

microbial load between cooked burger patties in the different kiosks is accepted. This means that

the microbial load in the cooked burger patties in all three kiosks also had high numbers of

microbial load despite having been heated and cooked.

In a study done by Tavakoli and Riazipour (2008), it is possible for cooked foods to be

contaminated with coliforms and pathogenic bacteria including E.coli and S.aureus, as 50% of

the 216 samples examined showed to have coliform contamination. S.aureus and E.coli

contaminations were found in 14.2% and 12.6% of the examined samples respectively.

Table 4. One-factor ANOVA results of the difference in the microbial load in cooked burger
patties between the different kiosks.
Source of Variation SS df MS F P-value F crit
Between Groups 3.12E+13 2 1.56E+13 0.763 0.507 5.143
Within Groups 1.23E+14 6 2.04E+13

Total 1.54E+14 8

To detect significant difference of microbial load between uncooked ground meat patties

in the different kiosks.

The data in Table 5 shows the summary of the results of the microbial load between

uncooked burger patties in the different kiosks from the 1:10000 dilution. The microbial load in
 35
the 1:10000 dilution for the three replications were added and the average and variance were

computed.

Table 5. Summary of results of the microbial load in uncooked burger patties between the
different kiosks.
Groups Count Sum Average Variance

Kiosk 1 3 13740000 4580000 6.4E+12

Kiosk 2 3 6973333 2324444 2.2E+12

Kiosk 3 3 10733333 3577778 1.64E+13

The data in Table 6 shows that no significant difference exists among the three kiosks

which serve as the sources of the uncooked burger patties. This is indicated in the p value of

0.652 which is greater than the level of significance at 0.05 which is also confirmed by the

critical value of 5.143 which is greater than the computed or the 0.459 F statistics.

Since the p value is greater than the level of significance, the null hypothesis for the

microbial load in uncooked burger patties between the different kiosks is accepted. The fact that

microorganisms were still present even in the cooked burger patties means that presence of

microorganisms in the uncooked burger patties were already expected as well. Uncooked burger

patties are, first and foremost, exposed to more environmental factors and with no application of

means to kill the microorganisms present in it, the microbial load in the uncooked burger patties

shows higher numbers of microorganisms present.

 36
Table 6. One-factor ANOVA results of the difference in the microbial load in uncooked burger
patties between the different kiosks.
Source of Variation SS df MS F P-value F crit
Between Groups 7.66E+12 2 3.83E+12 0.459 0.652 5.143
Within Groups 5.01E+13 6 8.35E+12
Total 5.78E+13 8

To detect potentially pathogenic bacteria that can cause infection in the gastrointestinal

tract, such as Enterohemorrhagic Escherichia coli and Salmonella spp., between cooked and

uncooked ground meat patties in the different kiosks.

The presence of E. coli in burger patties can be associated with the event that transpired

in Washington State on January 1993, wherein burger patties at a fast-food restaurant were

linked to undercooked ground beef and led to the event of an E. coli O157:H7 outbreak (Shefer

et al., 1996).

The data in Table 7 shows whether blue-black colonies with green metallic sheen grew

on EMB media using the 1:10000 dilution. The table shows that for the cooked burger patties,

three (3) samples were positive for blue-black colonies with green metallic sheen in the third

replication, and there is one (1) sample positive for blue-black colonies with green metallic sheen

also in the third replication. The fact that blue-black colonies with green metallic sheen,

indicative for growth of E. coli in EMB media, only occurred during the third replication meant

that at some point in the process, there must have been a factor for the inconsistent result since

there were no growth of blue-black colonies with green metallic sheen in the first and second

replications.
 37
Table 7. Distribution of E. coli O157:H7 on EMB media of cooked and uncooked burger patties
(1:10000 dilution)
COOKED UNCOOKED
KIOSK 1 S1 - -
S2 - + in the 3rd replication
S3 - -
KIOSK 2 S1 + in the 3rd replication -
S2 - -
S3 + in the 3rd replication -
KIOSK 3 S1 - -
S2 + in the 3rd replication -
S3 - -
(-): no growth of blue-black colonies with green metallic sheen on EMB
(+): growth of blue-black colonies with green metallic sheen on EMB

The researchers stated in the scope and limitations that certain factors such as the

temperature in which the burgers were cooked and the equipments used for the processing of the

patties are no longer within the control of the researchers. Other factors including the length of

time the sample has been stored before they were cooked or processed may also be the reason

why there were positive growth for E. coli during the third replication. Figure 6 shows the

growth of blue-black colonies with green metallic sheen on EMB media from the 1:10000

dilution.
 38

Figure 6. Growth of blue-black colonies with green metallic sheen on EMB from 1:10000
dilution.

In line with the results in the EMB media, the plates that had blue-black colonies with

green metallic sheen were subcultured to SMAC media. The data in Table 8 shows that the

colonies that grew in the SMAC media were all pink colonies. Therefore, the researchers

concluded that even though the colonies that grew were E. coli colonies, they were not classified

as E. coli O157:H7 since E. coli O157:H7 grow as colorless colonies in S-MAC medium.
 39
Table 8. Distribution of E. coli O157:H7 on SMAC media of cooked and uncooked burger
patties (1:10000 dilution).
COOKED UNCOOKED
KIOSK 1 S1
S2 -
S3
KIOSK 2 S1 -
S2
S3 -
KIOSK 3 S1
S2 -
S3
(-): no growth of colorless colonies on SMAC

Since, there were no colorless colonies that grew on the SMAC media, further tests for

the detection of E. coli O157:H7 were no longer continued by the researchers.

In a study done by Flores, Mendoza and Perez (2013) on the prevalence of E. coli

O157:H7 and other verotoxin-producing E. coli in raw, ground beef samples from wet markets in

Laguna, Philippines, they found out that the prevalence of E. coli O157:H7 in ground beef

collected from wet markets in Laguna was 0%. However, the prevalence of non-sorbitol-

fermenting, non-β-glucuronidase-producing E. coli in the collected samples was 5%. The

prevalence of verotoxin (Shiga-like toxin) - producing non-O157 E. coli in the beef samples was

5%.

For the detection of Salmonella spp. in the burger patties, the samples were first weighed

then homogenized in 225 ml Lactose broth solution. After the homogenization, the Lactose

broths were then incubated and checked for turbidity in the next 18-24 hours.

Table 9 shows the results for the detection of Salmonella spp. in burger patties. All the

Lactose broth in all three (3) replications were positive for turbidity, thus, further tests for the

detection of Salmonella spp. were continued. Aliquots were taken from the Lactose broth and

inoculated to XLD plates and TTB. After 18-24 hours, the XLD plates were checked for growth.
 40
Colonies that grew in the XLD media were yellow colonies, which are not indicative of any

Salmonella spp. since Salmonella spp. exhibit red colonies with black center.

Table 9. Distribution of Salmonella spp. on XLD media of cooked and uncooked burger patties
(1:10000 dilution).
COOKED UNCOOKED
KIOSK 1 S1 - -
S2 - -
S3 - -
KIOSK 2 S1 - -
S2 - -
S3 - -
KIOSK 3 S1 - -
S2 - -
S3 - -
(-) – no growth of red colonies with black center on XLD

Since there were no growth of red colonies or red colonies with black center for all three

(3) replications, further tests for the detection of Salmonella spp. were no longer continued.
 41
CHAPTER V

SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary

With the rising number of reported gastrointestinal diseases after consumption of

undercooked ground meat burger patties, the study seeks to detect the occurrence of E. coli

O157:H7 and Salmonella spp. in beef and pork burger patties sold by a number of food

establishments within the premises of Silliman University.

Three (3) kiosks were randomly selected and were identified to be selling burgers, which

were composed more of pork than of beef. Three (3) replications in each kiosk were done for the

study. To determine the microbial load in CFU/mL present in cooked and uncooked ground meat

patties, the samples were subjected to Aerobic Plate Count. For the bacterial identification,

routine culture for E. coli and routine culture for Salmonella spp. with a few modifications were

performed and was adapted from the Bacteriological Analytical Manual (BAM).

The results of the study showed no significant differences in the microbial load between

cooked and uncooked burger patties as well as no significant differences between cooked patties

and uncooked patties in the different kiosks. Escherichia coli O157:H7 and Salmonella spp. were

also not detected although other microorganisms such as other coliforms were found to be

present in both cooked and uncooked burger patties but were not further identified. No

significant difference in the microbial load between cooked and uncooked burger patties indicate

that the cooked burger patties contain low to high levels of contamination, therefore it is unsafe

for consumption especially by immunocompromised individuals.

 42
Conclusion

This study showed that there was no significant difference in the microbial load between

cooked and uncooked ground meat patties sold by food establishments within Silliman

University. There was also no significant difference in the microbial load of cooked burger

patties between the different kiosks, and uncooked burger patties between the different kiosks.

Though there was no significant difference of the microbial load between the uncooked and

cooked ground meat patties, the colony forming units per mL (CFU/mL) of some plates were

greater than 1.26 x105 CFU/mL which is under the unsatisfactory level of consumption based on

the NSW Food Authority (2009). The fact that the level of contamination in most of the samples

exceeds the satisfactory level of consumption means that the burger patties are unsafe for

consumption especially by immunocompromised individuals. There was no presence of E. coli

O157:H7 and Salmonella spp. in the burger patties sold by the different kiosks, though presence

of other coliforms were found but were not further identified.

In light of the foregoing findings and conclusions drawn from the study, it is highly

recommended that the succeeding researchers find a way to control the storage temperatures and

advise that all materials and equipments be sterilized for the processing of the burger patties that

will be collected as samples. The use of a “control” is also highly recommended to increase the

reliability and maintain standardization of the test procedures. The succeeding researchers may

also opt to use DNA barcodes in the identification of E. coli strains.