MODIFIED TEST FOR THE ACCELERATION IN

IDENTIFYING STAPHYLOCOCCUS AREUS USING

BRASSICA OLERACEA VAR. SABELLICA

(KALE) EXTRACT

A Thesis

Presented to the

Faculty and staff of the

PHINMA-University of Pangasinan

Arellano St., Dagupan City, Pangasinan

In partial

Fulfillment of the Requirements

For the Degree of Bachelor in Medical Laboratory Science

Submitted by:

Astadan, Loren Lem N.

Alacar, Jasmin Z.

Alba, Shalom

Ambeguia, Chelsea M.

Andongo, Sophia Vien C.

March 2018
 CHAPTER 1

Statement of the Problem

The study’s intention is to make a test that will speed-up the identification of

Staphylococcus aureus using Brassica oleracea var. sabellica (kale) extract.

This seeks to answer the following questions:

1. What is the effect of Brassica oleracea var. sabellica in accelerating coagulation?

2. How fast is the result compared to the original test?

3. What are the possible contributions and advantages of the modified test?
Rationale

Over the past years, studies from different organizations all over the world prove that

Staphylococcus aureus has been an important cause of nosocomial infections. Its various

infections may lead to relatively mild to life-threatening cases. (National Nosocomial Infections

Surveillance (NNIS) 2017). Further, NNIS nosocomial infection defined as “a localized or

systemic condition that results from adverse reaction to the presence of an infectious agent(s) or

its toxin(s) and that was not present or incubating at the time of admission to the hospital.”

(NNIS, 2017)

Although Staphylococcus aureus is a normal flora of the skin, it is considered as an

opportunistic pathogen and can invade human body and cause serious infections. A huge rate of

Staphylococcus aureus is now becoming an anti-biotic resistant bacteria, especially in areas such

as the Intensive Care Units (ICUs) compared to the other departments of the hospital.

(NNIS, 2017)

Researches found out that Asia is one of the regions with the highest prevalence rates of

healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and community-

associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the world. Most hospitals

in Asia are endemic for multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA),

with an estimated proportion from 28% (in Hong Kong and Indonesia) to >70% (in Korea)

among all clinical Staphylococcus aureus isolates in the early 2010s. (Chen, Huang 2014)

Staphylococcus aureus secretes coagulase enzymes which are not only virulence factors

but also essential criteria in differentiating it from other staphylococci like the Coagulase

Negative Staphylocci (CoNS). Several criteria like mannitol fermentation test, coagulase tests,
agglutination test, DNAse etc are proposed for discrimination of Staphylococcus aureus from

other staphylococci.

One of the most commonly used and reliable tests in the laboratory for detecting

Staphylococcus aureus and differentiating it from CoNS is the Coagulase Test which consists of

a series of tests that is notably somewhat time consuming. Enzyme coagulase is produced by

Staphylococcus aureus that converts soluble fibrinogen in plasma to insoluble fibrin.

(Gallo 2017)

There are two types of coagulase produced by Staphylococcus aureus. The bound

coagulase and the free coagulase (also known as Staphylocoagulase). Since there are two types

of coagulase produced, there are also two types of tests used. The slide coagulase test and the

tube coagulase test. The slide coagulase is a test that detects bound coagulase but not specific in

identifying Staphylococcus aureus while the tube coagulase test is used to detect the free

coagulase for a longer period of time and with higher specificity. (Aryal, 2015)

Knowing that Staphylococcus aureus is an agent in nosocomial infection there is a need

for rapid identification with high specificity for earlier diagnosis and treatment on the associated

disease.

Therefore, this research seeks to modify the said test, specifically, the tube coagulase

with the purpose of accelerating the identification of Staphylococcus aureus and to enhance its

specificity. In addition, the researchers aim to inform and encourage the laboratory personnels to

make use of this test for obtaining accurate results in a cost-effective, reliable and easier way.
 Moreover, students from PHINMA-University of Pangasinan conducted isolation and

identification of common bacterial nosocomial agents within hospitals in Pangasinan and found

out that Staphylococcus aureus is the second most frequently isolated bacteria in both public and

private hospitals next to Escherichia coli. (2017)

The Coagulation Cascade Theory

The classic theory of coagulation was described by Paul Morawitz in 1905. This model

described each clotting factor as a proenzyme that could be converted to an active enzyme

through a series of process. (cascade) It is the series of steps beginning with activation of the

intrinsic or extrinsic pathways of coagulation, or of one of the related alternative pathways, and

proceeding through the common pathway of coagulation to the formation of the fibrin clot. (

Raissa T. Guldam BSMT - IV)

It was said that Vitamin K serves as an essential cofactor for a carboxylase that catalyzes

carboxylation of glutamic acid residues on vitamin K-dependent proteins. And examples of these

Vitamin-K dependent proteins are factors II (prothrombin), VII, IX and X. Without vitamin K,

the carboxylation does not occur and the proteins that are synthesized are biologically inactive.

Hence absence of Vitamin K or insufficient amount of such would lead to slow coagulation

cascade or no cascade at all. (Previtali, 2011)

From the name itself, "Cascade", it is a series or sequence in which something is

successively passed on. And in our case we can be able to use this theory as a basis of our study.

On which we will focus our analysis on the ability of Vitamin K, which is a necessary participant

in the synthesis of several proteins that mediate coagulation, to accelerate the coagulation

process/cascade. ( Fiol, 2014)

Conceptual Framework

The two principal variables in an experiment are the independent and dependent

variables. An independent variable is the variable which must be controlled over, what can be

chosen and manipulated. A dependent variable is what researchers’ measure in the experiment

and what is affected during the experiment. The dependent variable responds to the independent

variable.

The researchers aim to determine the ability of Kale (Brassica oleracea var. sabellica)

leaves extract (independent variable) as a modified test for the acceleration in identifying

Staphylococcus aureus (dependent variable). By this, the researchers will be able to provide a

fast and natural way of obtaining accurate results in the field of medical laboratory science.
 Independent Variable Dependent Variable

Kale (Brassica oleracea var. Accelerated identification for

sabellica) leaves extract Staphylococcus aureus

Figure 1.1 Paradigm of the Study

Significance of study

As the field of Medical Laboratory Science advances together with much need for

specific and fast laboratory diagnosis, many laboratories are cumbered in dealing with this matter

especially with the limited resources and financial aspects relating to our country’s health

section. These situations give rise to the need to find new cost-effective and more accurate test of

providing service without sacrificing the quality and specificity. The will be benefited in the

study.

Medical Laboratory Science. The study can improve the isolation of Staphylococcus aureus

using some components of the Kale that can speed up the result in the free coagulase test. This

can further widen our knowledge about some factors that can speed up coagulation which can

also be used for isolating Staphylococcus aureus.

Medical Laboratory Research. The result of this study can motivate other researchers to

correlate future related studies. This can serve as their basis for their own researches. Given the

information, they can therefore know what are the other alternatives that can be used to

accelerate the identification of Staphylococcus aurues.

Medical Laboratory Practice. The study will benefit and help isolate the Staphylococcus

aureus in a short period of time knowing that this bacterium is a common nosocomial agent that

can cause serious infection and needs a rapid identification for early diagnosis and treatment on

the disease associated with Staphylococcus aureus which can be a great help among Medical

Laboratory Scientists if adapted in the laboratories.

Null Hypothesis

In this study, the researcher will test the null hypothesis at 0.05 level of significance.

Ho: There is no significant difference between the Kale (Brassica oleracea var. sabellica)

leaves extract and coagulase test in accelerating the identification of Staphylococcus aureus.

Scope and Delimitation

This study focus on using Brassica oleracea var. sabellica, commonly known as Kale,

for the enhanced or accelerated coagulation for the rapid identification of Staphylococcus aureus

using the tube coagulation test. The focus on the subject is on the high Vitamin K content, which

is a necessary participant in the synthesis of several proteins that mediate coagulation of the said

plant and also, there is a high content of other substances such as fiber, calcium and folate that is

of great importance in the coagulation cascade. The preparation of extracts and the accelerating

of coagulation procedure was performed at the Medical Technology Laboratory at the PHINMA-

University of Pangasinan. The study was conducted from the month of June to October at the

year 2018.
Definition of Terms

To provide clear and better understanding, the terms that will be frequently met in this

research are defined conceptually as follows:

Coagulase Test- is used to differentiate Staphylococcus aureus (positive)

from Coagulase Negative Staphylococcus (CONS). Coagulase is an enzyme produced by S.

aureus that converts (soluble) fibrinogen in plasma to (insoluble) fibrin. This is the test being

modified by the researchers. (Mahon, 2014)

Kale. Known as leaf cabbage; certain cultivars of cabbage (Brassica oleracea) grown for their

edible leaves is loaded with all sorts of beneficial compounds especially vitamin K with 1062.10

mcg content that is essential in the coagulation process when extracted. In this study, Kale is the

independent variable in accelerating the Identification of Staphylococcus aureus

(Ruhlman, 2016)

Nosocomial infection. A hospital-acquired infection (HAI), is an infection that is acquired in a

hospital or other health care facility. The most common pathogens that cause nosocomial

infections are Staphylococcus aureus, Pseudomonas aeruginosa, and E. coli. Some of the

common nosocomial infections are urinary tract infections, respiratory pneumonia, surgical site

wound infections, bacteremia, gastrointestinal and skin infections. (NNIS, 2017)

Staphylococcus aureus. A Gram-positive, round-shaped bacterium that is a member of the

Firmicutes, and it is a member of the normal flora of the body, frequently found in the nose,

respiratory tract, and on the skin. It is the subject being identified in the study. (Baorto, 2017)
 CHAPTER 2

This chapter contains statements enumerating the related literature, related studies and

synthesis of related studies. This chapter also gives the following information which is found to

be relevant to the study, involving Brassica oleracea var. sabellica (Kale) leaves extract as an

accelerating agent in identifying Staphylococcus aureus.

Related Literature (Foreign)

The staphylococci are gram-positive spherical cells, usually arranged in irregular clusters.

They grow readily on a variety of media and are active metabolically, fermenting many

carbohydrates and producing pigments that vary from white to deep yellow. The pathogenic

staphylococci often hemolyze bood and coagulate plasma. Some are members of the normal flora

of the skin and mucuos membranes of humans; others cause suppuration, abscess formation, a

variety of pyogenic infections, and even fatal septicaemia. (Jawetz et al, review of medical

microbiology 13th ed)

The term Staphylococcus aureus means “Gloden Cluster Seed” and it is also called as

“golden staph”. This microorganism was first isolated in 1884 by the Scottish surgeon – Sir

Alexander Ogston from surgical abscesses. (Ogston, 1884) It was named Staphylococcus (the

clusters of grape-like organisms), after the Greek word ‘Staphyle’ meaning bunch of grapes and

‘coccus’ which means granules. The species name ‘aureus’ originated from the Latin word

‘aurum’ which refers to the golden colour produced by the organism when grown on solid media.

(Howard & Kloos, 1987)

 Staphylococcus aureus remains one of the most significant pathogens causing disease in

animals and human and methicillin-resistant Staphylococcus aureus (MRSA) is ranked among

the most important and common pathogen resistant to multiple antibiotics all over the world. It is

surprising that a bacterium with such great potential for virulence as Staphylococcus aureus is a

common, intimate human associate. The microbe is present in most environments frequented by

humans and is readily isolated from fomites. Colonization of some infants begins within hours

after birth and continues throughout life. The carries rate for normal healthy adults varies

anywhere from 20% to 60%, and the pathogen tends to be harbored intermittently rather than

chronically. Carriage occurs mostly in the anterior nares (nostrils) and, to a lesser extent, in the

skin, nasopharynx, and intestine. Usually this colonization is not associated with symptoms, nor

does it ordinarily lead to disease in carriers or their contacts. Circumstances that predispose an

individual to infection include poor hygiene and nutrition, tissue injury, pre-existing primary

infections, diabetes mellitus, and immunodeficiency states. Staphylococcus infections in the

newborn nursery and surgical wards are the third most common nosocomial infection. The so-

called “hospital strains” can readily spread in an epidemic pattern within and outside the

hospital. (Talaro, Foundations in microbiology)

A serious concern has arisen from the increase in community infections by strains of

Staphylococcus aureus called MRSA (methicillin resistant Staphylococcus aureus). Several

outbreaks have been reported in prison inmates, athletes, and school children. The infections are

spread by contact with skin lesions, and have proved to be very difficult to treat and control.

(Talaro, Foundations in microbiology)

Pathogenic Staphylococcus aureus typically produce coagulase, an enzyme that

coagulates plasma and blood. The precise importance of coagulase to the disease process remains
uncertain. It may be that coagulase causes fibrin to be deposited around staphylococcus cells.

Fibrin can stop the action of host defenses such as phagocytosis, or it may promote

staphylococcus adherence to tissues. Because 97% of all human isolates of Staphylococcus

aureus produce this enzyme, its presence is considered the most diagnostic species characteristic.

An enzyme that appears to promote invasion is hyaluronidase, or the “spreading factor”, which

digests DNA (DNAse); and lipases that help bacteria colonize oily skin surfaces. Enzymes that

inactivate penicillin (penicillinase) and other antimicrobial drugs are produced by a majority of

strains, giving them multiple resistance. (Talaro, Foundations in microbiology)

Staphylococcus aureus is a common cause of skin (impetigo, boils, carbuncles, and

folliculitis), soft tissue (cellulitis), bone (osteomyelitis), and joint (septic arthritis) infections.

This species of Staphylococcus can also cause a number of toxin-mediated human illnesses such

as food poisoning, scalded skin syndrome, and toxic shock syndrome. There are several other

staphylococcal species that colonize the human. These species of Staphylococcus are less

commonly associated with human disease but are frequent contaminants in samples taken from

skin and soft tissue lesions. The coagulase test is one way to differentiate the highly

pathogenic S. aureus from the other less pathogenic staphylococcal species on the human

body. S. aureus is a coagulase-positive organism whereas all the other staphylococcal species

that colonize humans are coagulase negative. (Berke and Tilton, lin. Microbiol. 23:916–919)

Coagulase test is used to differentiate Staphylococcus aureus (positive) which produce

the enzyme coagulase, from S. epidermis and S. saprophyticus (negative) which do not produce

coagulase. i.e Coagulase Negative Staphylococcus (CONS). Coagulase is an enzyme-like protein

and causes plasma to clot by converting fibrinogen to fibrin. Staphylococcus aureus produces

two forms of coagulase: bound and free.

Bound coagulase (clumping factor) is bound to the bacterial cell wall and reacts directly

with fibrinogen. This results in an alternation of fibrinogen so that it precipitates on the

staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma.

This doesn’t require coagulase-reacting factor. Free coagulase involves the activation of

plasma coagulase-reacting factor (CRP), which is a modified or derived thrombin molecule, to

from a coagulase-CRP complex. This complex in turn reacts with fibrinogen to produce the

fibrin clot.

There are two tests that tend to identify bound coagulase and free coagulase. First is the

Slide Coagulase Test. This method measures bound coagulase. The bound coagulase is also

known as clumping factor. It cross-links the α and β chain of fibrinogen in plasma to form fibrin

clot that deposits on the cell wall. As a result, individual coccus sticks to each other and

clumping is observed. Second is the Tube Coagulase Test.

This method helps to measure free coagulase. The free coagulase secreted by S.aureus

reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin.

This converts fibrinogen to fibrin resulting in clotting of plasma. Blood clots are formed through

a series of chemical reactions in your body. Vitamin K is essential for those reactions. Vitamin K

is known as the clotting vitamin, because without it, blood would not clot. It increases the

chemical reactions in your body needed for your blood to clot. The more Vitamin K you take the

more chemical reactions your body makes for your blood to clot. Hence your blood gets

"thicker". Also, some studies suggest that it helps maintain strong bones in the elderly.
 Vitamin K refers to a group of fat-soluble vitamins that play a crucial role in blood

clotting. They act as a co-factor for seven vitamin K-dependent clotting factors, because without

vitamin K, your blood is unable to clot. You get vitamin K from a variety of foods in your diet.

Rich sources include liver, turnip greens, broccoli, kale, cabbage and asparagus. Vitamin K

causes the blood to coagulate, thereby reducing the risk of bleeding. This effect may interact

with certain medications. Vitamin K is a fat-soluble vitamin stored in your fat and liver tissue.

Green leafy vegetables, such as kale and spinach, contain the largest amounts of vitamin K. Do

not eat large amounts of these foods if you take anticoagulant medications; high vitamin K may

disrupt the medication's efficacy. Men need about 19 mcg per day, while women only need 90

mcg. Children need more as they get older. (Renee, 2017)

The only known unequivocal biological role of vitamin K is as a cofactor for

an enzyme that enables specific proteins to bind calcium. The ability to bind calcium ions (Ca2+)

is required for the activation of the seven vitamin K-dependent blood clotting (‘coagulation’)

factors (e.g., prothrombin), or proteins, in the series of events that stop bleeding through clot

formation (‘coagulation cascade’). Vitamin K-dependent coagulation factors are synthesized in

the liver. Consequently, severe liver disease results in lower blood levels of vitamin K-dependent

clotting factors and an increased risk of uncontrolled bleeding (‘haemorrhage’). (Dr Peter Engel

in 2010, reviewed and updated by Dr Szabolcs Peter on 2017)

Vitamin K is produced by the bacteria in your intestines, and it is also in vitamin and

nutritional supplements. Your body uses vitamin K to produce some of the clotting factors that

helps blood clot. Vitamin K is a naturally occurring vitamin. Vitamin K is primarily found in

leafy green vegetables such as spinach, broccoli, and lettuce, and enters your body when you eat

these foods. (Laura Earl, RN, BSN, CACP

 It was said that kale is an excellent source of vitamin K, vitamin C, vitamin A,

manganese, and copper; a very good source of vitamin B6, fiber, calcium, potassium, vitamin E,

and vitamin B2; and a good source of iron, magnesium vitamin B1, omega-3 fats, phosphorus,

protein, folate, and vitamin B3. (Angeloni, Leoncini, Malaguti, et al.)

Kale is a leafy vegetable in the Brassica or cole crop family. It is usually grouped into the

"Cooking Greens" category with collards, mustard and Swiss chard, but it is actually more of a

non-heading cabbage, although much easier to grow than cabbage. The leaves grow from a

central stem that elongates as it grows. Kale is a powerhouse of nutrients and can be used as

young, tender leaves or fully grown. Kale can be grown as a cut and come again vegetable, so a

few plants may be all you need. The plants can be quite ornamental, with leaves that can be curly

or tagged, purple or shades of green. It is considered a cool season vegetables and can handle

some frost, when mature. Boiling raw kale diminishes most of these nutrients except for

vitamin K. (Iannotti, 2017)

It is one of the excellent vegetable sources for vitamin-K; 100 g provides about 587% of

recommended intake. Vitamin-K has a potential role in bone health through promoting

osteoblastic (bone formation and strengthening) activity. Adequate vitamin-K levels in the diet

help limiting neuronal damage in the brain; thus, has an established role in the treatment of

patients who have Alzheimer's disease. (Nutrition and You.com)

Related Literature (Local)

Staphylococcus aureus, which is present in human skin and nostrils, was one of the

earlier pathogenic bacteria that became resistant to penicillin. The first resistant strain was
recorded in 1947, four years after penicillin-based antibiotics started being mass-produced. At

the time, the antibiotic of choice was named methicillin, so the strain was termed methicillin-

resistant staphylococcus aureus. The bacterium which is present in the human skin and nostrils

can enter the body through a cut or puncture in the skin. This can cause infections such as

impetigo, boils, abscesses, folliculitis and cellulitis. Some more severe infections develop into

sepsis, septicemia, toxic shock syndrome, urinary tract infection and pneumonia.

MRSA infection is categorized as healthcare associated when it occurs in a hospital, or

community associated when acquired elsewhere. At risk from MRSA infection are people with

weak immune systems such as HIV/AIDS, lupus or cancer sufferers, transplant recipients and

severe asthmatics. Also at risk are diabetics, intravenous drug users, young children, elderly and

those in crowded places such as college students living in dormitories, health care facility

residents or workers, prison inmates, military recruits in training, occupants of homeless shelters

or gym users.

The Philippines reported the first case of MRSA at the Philippine General Hospital in

1987. The following year, the Department of Health and the Research Institute for Tropical

Medicine began the Antimicrobial Resistance Surveillance Program, which monitors resistance

of selected bacteria to specific antimicrobials in identified hospitals nationwide. Based on the

annual ARSP reports, MRSA rates rose from less than 20% of infection cases in 1988 to 45% in

2009. (Pascual, 2014)

Dr. Gigi Claveria, Pfizer Philippines senior medical manager. Many Filipinos are still not aware

of a serious health threat that doctors are always on the lookout for. MRSA or Methicillin

Resistant Staphylococcus aureus is one of the types of drug-resistant bacteria, and cases of
infection from this so-called “superbug” have been slowly rising in the country. This was the

warning of the Philippine Society for Microbiology and Infectious Diseases (PSMID) at World

MRSA Day recently.

The latest data from the Research Institute of Tropical Medicine (RITM), which monitors

MRSA infection cases in various hospitals across the country, showed that the resistance rate of

MRSA in antibiotics increased slightly to 62.6% in 2015 from 60.3% in 2014. “Staph” is

commonly present in different parts of the body, such as the nose and sometimes on the skin, and

yet it is also potentially one of the most dangerous bacteria. There are strains that are highly

resistant to antibiotics, called MRSA, which may cause different types of infections.

Otherwise healthy individuals are vulnerable to MRSA infection. This form, called

community-associated MRSA, often begins as a painful skin boils and is usually spread through

skin-to-skin contact. Reported cases of this infection often involve athletes, childcare workers,

people who have been exposed in hospitals or other health care settings, and those who live in

crowded conditions such as prisoners. Data from RITM’s monitoring program called

Antimicrobial Resistance Surveillance Program (ARSP) showed that for 2015, 85% of MRSA

isolates were presumptively community associated. Since then, “we’ve been noticing that there is

increasing incidence of community acquired MRSA,” said Philippine Society for Microbiology

and Infectious Diseases (PSMID) president Dr. Mari Rose delos Reyes. “Before, MRSA patients

were those in the hospital. Now, they are from everywhere.”

MRSA infection is carefully treated with antibiotics such as clindamycin, trimethoprim-

sulfamethozaxole, doxycycline, minocycline, or linezolid. Glycopeptides have been the usual

therapy of severe MRSA infections, but strains resistant to such antibiotic have emerged. Despite
the seeming invincibility of MRSA, infectious disease specialist and PSMID past President Dr.

Marie Yvette Barez reminds the public of a very simple practice that can stop it in its tracks —

hand washing. Dr. Barez said that because our hands touch so many things every day, our 10

fingers are some of the dirtiest parts of our body. She added, “For MRSA patients, they should

be isolated and their caregivers should wear mask and gowns for protection.” “There are still so

many people who do not know what MRSA is or how to prevent its spread,” said Dr. Gigi

Claveria, pulmonologist and Pfizer Philippines senior medical manager. “That is why with this

new campaign, we want to encourage more people to ‘make a move’ and take care of their health

by learning more about how they can prevent MRSA. (The Philippine Star, 2016)

Related Studies (Foreign)

Rapid Identification of Staphylococcus aureus from Blood Culture

Bottles by a Classic 2-Hour Tube Coagulase Test

The rapid, reliable identification of Staphylococcus aureus from positive blood cultures provides

important information. While multiple methodologies for detection of S. aureus from blood

culture broths exist, none is satisfactory. Immunologic tests have shown varied sensitivities, the

thermo nuclease test, while sensitive, are not practical for routine use, and probe tests are

expensive. Few studies have addressed using the tube coagulase test (TCT). This study compared

two immunologic methods, the Staph Latex kit (Remel Laboratories) and the Staphaurex kit

(Wellcome Diagnostics), with a rabbit plasma TCT (Difco Laboratories) to identify S. aureus
within 2 h directly from blood culture broths and pelleted supernatants from BACTEC (Johnston

Laboratories) bottles. One hundred twelve unique clinical blood culture isolates consistent with a

Gram stain for staphylococci and 68 negative blood culture bottles seeded with a variety of

gram-positive organisms were evaluated. Sensitivity and specificity among clinical specimens

for the 2-h TCT were 79.5 and 100%, respectively. Sensitivities for the immunologic methods

were 12.8 and 10.2% for the Staphaurex and Remel Staph Latex, respectively, and specificities

for both were 100%. These results contradict previously reported results for both immunologic

and TCT methods and dictate that a specific as well as a sensitive method be employed. The 2-h

TCT was found to be a cost-effective, reliable, and rapid method for identifying S. aureus from

positive blood cultures. J. Clin. Microbiol. 1995, PCR Assay for Detection of Staphylococcus

aureus in Fresh Lettuce (Lactuca sativa)

The aim was to determine the incidence of S. aureus in fresh lettuce by PCR in order to

enhance the efficiency for detection and identification process. For coa gene, the temperature

gradient showed that 56°C was the optimal annealing temperature (Ta) for oligonucleotides,

showing an adequate specificity for the detection of S. aureus.

The Ta is defined as the highest temperature where the optimal aligning and

amplification occur; this parameter is crucial for the standardization of the method because a low

Ta can cause nonspecific amplification, giving undesired PCR products; this is when two or

more bands are observed in gel electrophoresis. In this study, the primers features and the correct

156 Frontiers in Staphylococcus aureus design lead us to obtain a good and specific

amplification in a range of 56–60°C. Likewise, a high Ta can cause a low or non‐amplification,

reducing the possibility to anneal; for this reason, an optimization of priming temperature is

necessary. Additionally, annealing was satisfactory at low DNA concentrations (up to 0.5 pg/μl)
showing adequate sensitivity. Isolated from lettuce samples were confirmed by amplification of

the 674 bp fragment.

For the strategy with the 16S an optimal annealing temperature of 54°C was established

for a fragment of approximately 1400 bp; isolates 1 and 2 were aligned in the same clade as the

positive control (ATCC 11632) strain. Clinical animal isolates reported at NCBI D83357.1,

D83355.1 and isolated from human throats suffering clinical infections JN315147.1,

JN390832.1, JN390831.1, JN315154.1, JN315153.1, JN315151.1, JN315150.1, and JN315149.1

show that isolates 1 and 2 are potentially dangerous if the vegetable is not properly sanitized

before consuming.

Low incidence of S. aureus is directly related to good manufacturing practice of packing

companies, mainly because the exposure time of the product in contact with the exterior is very

short. Likewise, the product is never in direct contact with the staff due to the use of hairnets,

gloves, face masks, aprons, and boots, as well as all staff washing and disinfecting their hands

before entering work and after toileting.

The bacterial counts found in this study were below the health limit of 102–103 CFU g‐1

of S. aureus in food set by the Codex Alimentarius, stabilizing a good quality of lettuce with

respect to this pathogen. A study by Viswanathan and Kaur reports the presence of S. aureus in

23% of a total of 120 samples from various vegetables in India. This incidence is attributed to

postharvest and human contamination due to the management of the foods. These results make

evident the permanence of the pathogen in this food group, the proper handling of Mexican

producers, and the safety of their food. The molecular techniques used in this study are suitable

for the identification of S. aureus isolated from lettuce, increasing our capability of detecting this
pathogen by improving the process and increasing the efficiency, contributing to the safety of

this vegetable. Andrés F. Chávez‐Almanza et al

Evaluation of the antimicrobial activities of ultra-sonicated spinach leaf extracts using

RAPD markers and electron microscopy. Spinach (Spinacia oleracea L.) leaves represent an

important dietary source of nutrients, antioxidants, and antimicrobials. As such, spinach leaves

play an important role in health and have been used in the treatment of human diseases since

ancient times. Here the aims were to optimize the extraction methods for recovering

antimicrobial substances of spinach leaves, determine the minimum inhibitory concentrations

(MICs) of the antimicrobial substances against Escherichia coli and Staphylococcus aureus and

finally, evaluate the effects of spinach leaves’ antimicrobials on bacterial DNA using central

composite face centered methods (CCFC). The effect of the extracts on both Gram positive and

Gram negative bacterial models were examined by scanning electron microscopy (SEM) and

random amplification of polymorphic (bacterial) DNA (RAPD). The optimal extraction

conditions were at 45°C, ultrasound power of 44% and an extraction time of 23 min. The spinach

extracts exhibited antimicrobial activities against both bacteria with MICs in the 60-100 mg/ml

range. Interestingly, SEM showed that treated bacterial cells appear damaged with a reduction in

cell number. RAPD analysis of genomic DNA showed that the number and sizes of amplicons

were decreased by treatments. Based on these results, it was inferred that spinach leaves extracts

exerts bactericidal activities by both inducing mutations in DNA and by causing cell wall

disruptions.

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-

negative staphylococci from blood culture material: a comparison of six bacterial DNA

extraction methods
 Staphylococcus aureus is a pathogen which can cause both hospital- and community-

associated infectious diseases, ranging from minor skin infections to endocarditis, bacteremia,

sepsis and septic shock. Sepsis can result in high morbidity and mortality. In the United States,

bloodstream infections develop in approximately 250,000 people annually. In the Netherlands,

the incidence of patients admitted to the intensive care unit (ICU) with severe sepsis is in the

range of around 8,643 ± 929 per year. Currently, blood culture is the gold standard for the

identification of pathogens from suspected bacterial sepsis patients. Unfortunately, blood culture

is time-consuming, taking at least 24–72 h for the final determination of the bacteria causing the

disease. Staphylococci are the most common Gram-positive organisms in blood cultures.

Differentiating S. aureus from coagulase-negative staphylococci (CNS) is important, because

sepsis with S. aureus is common and virulent, with mortality rates in the range of 20–30%. CNS

are often considered as being contaminants in blood cultures due to the fact that these species are

members of the normal skin flora and mucous membranes, and can contaminate the sample when

it is taken. However, it is known that CNS infections are increasingly recognized as clinically

relevant infections and confirmation on the presence of these species in blood culture is,

therefore, important (reviewed in). Several molecular methods for the rapid and accurate

detection of bacteria from positive blood culture material have been described, including

(commercial) real-time polymerase chain reaction (PCR)-based diagnostic tests, fluorescence in

situ hybridization, matrix-assisted laser desorption ionization time-of-flight mass spectrometry

(MALDI-TOF-MS) and also DNA micro-arrays. However, all of these techniques are used on

positive blood culture material.

Reduction in the time to obtaining results can be achieved by applying molecular

methods either directly on whole blood or on blood culture material with reduced incubation
times. Ideally, usage of whole blood is preferred but the techniques that are now available are

often not sensitive enough, clinically, as has been shown by others investigating a commercial

real-time PCR test currently available. Blood culture materials are known to contain inhibiting

factors which can reduce detection in a sensitive real-time PCR. It is, therefore, important to

include a good isolation method in the molecular diagnostic strategy, which is able to efficiently

remove inhibiting factors and one which still allows sensitive DNA detection by PCR.

In this study we compared six different, both manual and automated, bacterial DNA

isolation methods for two commonly used blood culture systems, i.e. BACTEC (Becton

Dickinson) and BacT/ALERT (bioMérieux), to be able to find the most sensitive bacterial DNA

isolation method. Additionally, we investigated the occurrence of inhibition in PCR

amplification after DNA isolation. A sensitive real-time PCR assay was designed to be able to

detect staphylococci and to differentiate S. aureus from CNS (Loonen et al., manuscript

submitted). Subsequently, this real-time PCR was used in combination with the optimal DNA

isolation method to investigate the level of time reduction to identify staphylococci from blood

culture material. The results were compared with conventional blood culture techniques used in

diagnostic laboratories. (Eur J Clin Microbiol Infect Dis. 2011)

Related Studies (Local)

The clinical and epidemiologic profile of community-associated methicillin-resistant

staphylococcus aureus infection among pediatric patients admitted at the Philippine General

Hospital.
 Several studies have reported increasing prevalence of methicillin-resistant

Staphylococcus aureus (MRSA) infection among patients with no predisposing factors. This

paper aims to determine the clinical and epidemiologic profile of community-associated MRSA

(CA-MRSA) infection among children admitted at UP-PGH.

A retrospective review of the medical records of patients 0 to 18 years old with S. aureus

isolate admitted at University of the Philippines-Philippine General Hospital (UP PGH) from

January 1, 2007 to December 31, 2008 was conducted. S. aureus isolates were classified as

methicillin-susceptible S. aureus (MSSA), CA-MRSA or healthcare-associated MRSA (HA-

MRSA). Risk factors for MRSA acquisition were identified. Demographic data, site of infection,

outcome, and antibiotic susceptibility patterns were compared.

S. aureus was isolated in 382 children. Medical records of 219 (57.33%) patients were

available for review. Of the 219 patients, 40.64% had MSSA, 15.07% had CA-MRSA, and

44.3% had HA-MRSA isolates. The prevalence of CA-MRSA is seven per 1000 admissions.

There was no statistical difference between the age, sex, outcome and the site of infection among

the three groups. The most common source of isolates was exudates, followed by blood. There

were statistically significant differences in the resistance patterns of S. aureus isolates, with

MSSA and CA-MRSA having lower resistance rates (<10%) as compared to HAMRSA (>40%)

and non-beta lactam antibiotics such as tetracycline, clindamycin, cotrimoxazole, gentamicin and

vancomycin.

This study showed that MRSA infection is no longer limited to patients with predisposing

factors. The type of S. aureus infection cannot be predicted based on clinical and demographic

profile of patients. Based on the susceptibility patterns in this study, CA-MRSA may be treated
with tetracycline, clindamycin, cotrimoxazole, gentamicin and vancomycin. (Aragon, MD et al,

2011)

Methicillin-resistant Staphylococcus aureus Hand Infections at UP-PGH: A Retrospective

Study from 2005-2010. Infections of the hand can result in profound morbidity, including

stiffness, contracture, and amputation, if not recognized early and given the appropriate

antibiotics. Factors that influence the outcome of infection include location of infection,

causative organism, timing of treatment, adequacy of surgical drainage, efficacy of antibiotic

therapy, health status and immunocompetence of the infected person. The most common

organism isolated from hand and other soft tissue infections is Staphylococcus aureus which

thrive as human skin flora. First-generation cephalosphorins have been traditionally the

cornerstone antibiotic treatment for acute hand infections.

Methicillin-resistant Staphylococcus aureus (MRSA) was first described in 1961, a short

period after the introduction of methicillin in 1959. Most reported cases were hospital acquired

(HA-MRSA). 6-8 Established risk factors for MRSA infection include recent hospitalization or

surgery, residence in a long-term care facility, dialysis, and indwelling percutaneous medical

devices and catheters.

By the mid-1990s, however, younger and otherwise healthy individuals were acquiring

MRSA infections despite not having contact with the hospital environment. This has started the

concept of community-acquired methicillin- resistant Staphylococcus aureus (CA-MRSA)

infections. This type of infection has been associated with a high incidence in at-risk populations

and settings, including extremes of age, contact sports, shared athletic equipment,

immunosupression, prisons, daycare centers, households, schools, and athletic or military

facilities.6,8 In recent years, however, CA-MRSA has been shown to have increasing prevalence

in soft tissue infections. In particular, CA-MRSA infections of the hand have been increasing

since the first case reports in 2000 have been described.

At the researcher’s institute, a study done by the Hospital Infection Control Unit (HICU)

and Bacteriology Section showed that the overall MRSA incidence is 51% in 2009 from 50% in

2008. The incidence of hand infections, however, was not specified.

The objectives of the present study were (1) to determine the incidence of MRSA among

admitted hand patients at UP-PGH from 2005 to 2010; and (2) to determine the antibiotic

susceptibility pattern. Specifically, (1) to determine the most common types of hand infections;

(2) to determine the most common sites of involvement; and (3) to determine the most common

location of involvement. (Bautista1, 2011)

 CHAPTER 3

Research Methodology

This chapter provides the materials and methods that will be used in respectively in this

research. This includes the research design, locale of the study, sources of data, data gathering

procedure and tools for data analysis.

Research Design. The method to be used in this research is quantitative-experimental. It seeks to

determine the effect of Kale (Brassica oleracea var. sabellica) leaves extract as an accelerating

agent in identifying Staphylococcus aureus. An experimental method of research will be applied

wherein a controlled environment with manipulated treatments termed as independent or

experimental variable may be applied in the sample group.

Locale of the Study. The preparation of Kale (Brassica oleracea var. sabellica) leaves extract

and the experimentation itself will be performed indoor at PHINMA-University of Pangasinan

Medical Technology Laboratory, Dagupan City, Pangasinan. The isolation of Staphylococcus

aureus will be conducted from public hospitals such as Pangasinan Provincial Hospital and

Region 1 Medical Center, Dagupan City

Sources of Data. The Brassica oleracea var. sabellica (Kale) that we will be using is bought at

the Locale City of Baguio, as for the population of the specimen used in the course of the

experiment, we used a healthy kale which is cooked, boiled and drained without salt. (Reason for

this is for easier way of obtaining the extract needed for the experimental research for it has a

higher content of vitamin K compared to a raw kale) The Staphylococcus aureus will be isolated

at public hospitals specifically at the Pangasinan Public Hospital (PPH) and Region 1 Medical

Center (R1MC) for it is a well-known nosocomial agent.

Data Gathering Procedure.

Tools for Data Analysis.