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Isolation and Identification of

Staphylococcus aureus from environmental factors.

Priyangka Ramiah

Made Agus Hendrayana

Program Studi Pendidikan Dokter

Fakultas Kedokteran Universitas Udayana

Denpasar

2017

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CONTENT

Report cover…………………………………………………………….................0

Content…………………………………………………………………….............1

CHAPTER I INTRODUCTION

1.1. Background………………………………………………………………....2

1.2. Problem identification………………………………………………………...4

1.3. Aims…………………………………………………………………………..5

1.4. Benefits……………………………………………………………………….5

CHAPTER II LITERATURE REVIEW

2.1. What is Staphylococcus aureus? ..................................................................6-9

2.2. What are the environmental factors that Staphylococcus aureus is

found in ?...................................................................................................10-11

2.3. How is Staphylococcus aureus identified, isolated and diagnosed……...11-19

CHAPTER III CONCLUSION……………………………………………….…20

References……………………………………………………………….……21,22

Jurnal Asli

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CHAPTER I

INTRODUCTION

1.1 Background

Staphylococcus is gram-positive cocci that are microscopically observed as

individual organisms, in pairs, and in irregular clusters. Staphylococcus aureus is

a round-shaped bacterium and is frequently found in the nose, respiratory tract,

and on the skin. These bacteria are present in up to 25 percent of healthy people

and are common among those with skin, eye, nose, or throat infections.

Sir Alexander Ogston a Scottish surgeon who first observed staphylococci and

discovered the major cause of pus. He found the bacterium during an examination

of a pus sample removed from the leg of a man in his private laboratory. His

hypothesis concluded that acute abscesses were caused by micrococci. After

injecting pus from acute abscesses into guinea pigs and mice, he demonstrated

that new abscesses were formed, following the signs of septicaemia. He stained

the cocci using methylaniline which allowed him to study the different types of

cocci using the form of growth. He reported his observation at the Ninth Surgical

Congress in Berlin in 1880. In 1882 Ogston described the cocci, Staphylococcus,

as being grape-like clusters which is called as staphyle in Greek.

In 1884 Anton J. Rosenbach, a German surgeon, isolated two pigmented

varieties in pure culture, and provided the first taxonomic description of the new

genus, dividing it into Staphylococcus aureus, which comes from the word aurum

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known as gold in latin, and Staphylococcus albus which is known as white in latin

and now called as epidermidis. (A Brief History of Staph, 2017)

In 1930s a coagulation test enables scientist to detect a plasma-coagulating

enzyme secreted by S.aureus, still the most common cause of wound infections in

hospitals. In 1940s the mortality rate of S.aureus is reported as 81% in a Boston

hospital. That same year, a British policeman seriously ill with S. aureus is cured

by penicillin. Benzyl penicillin cures many staph infections. But this leads to laps

in antiseptic and aseptic protocols. Later on penicillin-resistant staph outbreaks

occurred. In 1959, penicillin was no longer able to control staph infections.

(A Brief History of Staph, 2017)

Methicillin was introduced in Europe and has short-life uses as the bacteria

evolve to resist it. 1961, Methicillin-resistant S.aureus now known as MRSA

was detected in a British hospital. This drug becomes very known in Europe,

Australia and United States. In 1970s European countries establish infection-

control regulations. MRSA rate drops while increasing in United States and

Britain. Data of Staphylococcus aureus carriage in Indonesian hospitals are rare.

Therefore, the epidemiology of S. aureus among surgery patients in three

academic hospitals in Indonesia was studied. Total, 366 of 1,502 (24.4%) patients

carried S.aureus. (A Brief History of Staph, 2017)

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1.2 Problem identification

Problems identified in the writing of this paper are:

1. What is Staphylococcus aureus?

2. What are the environmental factors that Staphylococcus aureus is found

in?

3. How is Staphylococcus aureus identified, isolated and diagnosed.

1.3 Aims

Aims of writing this paper are:

1. To understand Staphylococcus aureus.

2. To understand how Staphylococcus aureus effects the environmental

factors.

3. To identify and isolate Staphylococcus aureus.

1.4 Benefits

This paper is expected to benefit some parties, such as:

1. Students can be used as reference or guidance for assignments regarding

Staphylococcus aureus.

2. Readers can enhance knowledge and understanding on Staphylococcus

aureus.

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CHAPTER II

LITERATURE REVIEW

2.1 What is Staphylococcus aureus?

As explained previously, Staphylococcus aureus according to the biological

characteristics are gram-positive and a round-shaped bacterium and is frequently

found in the nose, respiratory tract, and on the skin. It can grow without oxygen

and is often positive for catalase and nitrate reduction. S. aureus is a common

cause of skin infections including abscesses, respiratory infections therefore is not

always pathogenic. It is not motile and does not form spores. S.aureus reproduce

by binary fission and can cause skin and soft tissue infections particularly when

skin or mucosal barriers have been breached. S. aureus infection spreads through

contact with pus from an infected wound, skin-to-skin contact with an infected

person, and contact with objects used by infected person. Staphylococcus aureus

can live in the body for years undetected. Once symptoms begin to show, the host

is contagious for another two weeks and the overall illness lasts a few weeks. If

untreated though, the disease can lead to death. Staphylococcus aureus is a gram-

positive bacteria, which means that the cell wall of this bacteria consists of a very

thick peptidoglycan layer. They form spherical colonies in clusters and have no

flagella. Secretions are numerous, but include surface associated adhesins,

exoenzymes, and capsular polysaccharides. The capsule is responsible for

enhanced virulence of a mucoid strain. The central routes of glucose metabolism

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are the Embden-Meyerhof-Parnas (EMP) pathway and the pentose phosphate

cycle. (V Chan, 2006)

Lactate is the end product of anaerobic glucose metabolism and acetate and CO2

are the products of aerobic growth conditions. S. aureus can uptake a variety of

nutrients including glucose, mannose, mannitol, glucosamine, N-

acetylglucosamine, sucrose, lactose, galactose and beta-glucosides. (V Chan,

2006)

Domain: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Staphylococcaceae

Genus: Staphylococcus Rosenbach 1884

Species: S.aureus

(V Chan,2006)

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Picture1: Gram positive S.aureus under Picture 2: Staphylococcus aureus from

light microscope, microscopic view.

Magnification at 100x Magnification: 10,000x under scanning

electron micrograph (SEM).

(source: Janice Haney Carr, Matthew)

Golden yellow colonies

Picture 3: Staphylococcus aureus cultured from blood agar.

Pigment carotenoid moves through a pathway producing

the golden colour staphyloxanthin.

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2.2 What are the environmental factors that Staphylococcus aureus is

found in?

The environmental factors that are found in staphylococcus aureus is from

infected wound and skin, marine environment and contaminated food.

Staphylococcus aureus present in marine environments basically polluted

contaminated water that has been related to the number of bathers and may cause

diseases in skin, eye or ear. There are still many rural areas here in Bali,

Indonesia. Therefore all those people live in rural area bath and do all their daily

activities using the contaminated river or lake water. These people might even

use the contaminated water to drink and cook. (H. C. Thaker, 2012)

Wound and skin infections represent the invasion of tissues by

one or more species of microorganism. This infection triggers the body's immune

system, causes inflammation and tissue damage, and slows the healing process.

Staphylococcus aureus is the most dangerous of all of the many common

staphylococcal bacteria. These bacteria spread by having direct contact with an

infected person, by using a contaminated object. Skin infections are common, but

the bacteria can spread through the bloodstream and infect distant organs. Skin

infections may cause blisters, abscesses, and redness and swelling in the infected

area. Skin that may contain staphylococcus aureus are such as scalp and dry skin.

(H. C. Thaker, 2012)

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Staphylococcus aureus causes food poisoning when a food is

contaminated by not properly refrigerated such as milk and basically most of the

dairy products. Other sources of food contamination include the equipment and

surfaces on which food is prepared. These bacteria multiply quickly at room

temperature to produce a toxin that causes illness. It is killed by cooking and

pasteurization. Many food products including raw retail meat contains

staphylococcus aureus. (H. C. Thaker, 2012)

2.3 How is Staphylococcus aureus identified, isolated and diagnosed?

The principle that isolates are identified with a combination of colonial

appearance, serology and biochemical tests. If confirmation of identification is

required, the isolates should be sent to the Reference Laboratory. There are many

ways to identify Staphylococcus aureus :

A. Under a microscope.

Picture 4: Staphylococcus aureus under microscope.

Gram-positive. Cocci in grape-like clusters ( dark blue in colour)

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B. Culture

Baird Parker Agar (BPA)

A loopful of sample which is mixed with peptone water is streaked on BP agar

and incubated for 48 hours at 37°C. Characteristic appearance of jet black

colonies surrounded by a white halo were considered to be presumptive S. aureus

The pure cultures is streaked on Nutrient agar and incubated for 24 hours at 37°C

and were further characterized by biochemical tests. (H. C. Thaker, M. N.

Brahmbhatt, J. B. Nayak, 2012)

Blood Agar

Beta-hemolytic colonies of Staphylococcus aureus on sheep blood agar.

Cultivation 24 hours, aerobic atmosphere, 37°C. Yellow colored colonies of

Staphylococcus aureus on Tryptic Soy Agar. Carotenoid pigment staphyloxanthin

is responsible for the characteristic golden colour of S. aureus colonies. This

pigment acts as a virulence factor. Cultivation 24 hours in an aerobic atmosphere,

37°C. Colonies of Staphylococcus aureus seen with transmitted light. Cultivated

on Columbia agar with 5% defibrinated sheep blood, 24 hours in an aerobic

atmosphere, 37°C. Colonies are surrounded by a wide zone of beta-hemolysis.

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Picture 5 Picture 6

Picture 7

Picture 5: Beta haemolytic colonies of Staphylococcus Aureus.

Picture 6: Yellow coloured colonies of Staphylococcus aureus on tryptic soy agar.

Picture 7: Colonies of Staphylococcus aureus seen with transmitted light

(Source : micrbiologyinphotos.com)

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Mannitol Salt Agar (MSA)

Mannitol Salt Agar contains peptones and beef extract 1.0 gm, which supply

nitrogen, vitamins, minerals and amino acids . Contains pancreatic digest of

casein 5.0 gm, peptic digest of animal tissue 5.0 gm. The 7.5% concentration of

sodium chloride that supplies essential electrolytes for transport and osmotic

balance. D-Mannitol is the fermentable carbohydrate that leads to acid production.

Phenol red indicator to detect the fermentation which leads to acid production.

Coagulase positive in Staphylococcus aureus produce yellow colonies and a

surrounding yellow medium. Coagulase negative Staphylococcus aureus produce

red colonies and no color change of the phenol red indicator. Agar is the

solidifying agent. (Sagar Aryal,2016)

C. Biochemical Test

The coagulase test

Slide Test

Two drops of saline are put onto the slide. The two saline drops are emulsified

with the test organism using a wire loop, straight wire, or wooden stick. A drop of

plasma is placed on the inoculated saline drop corresponding to test, and mixed

well, then the slide is rocked gently for about 10 seconds. If 'positive',

macroscopic clumping would be observed in the plasma within 10 seconds, with

no clumping in the saline drop. If 'negative', no clumping will be observed. If the

slide coagulase test is negative, a tube test should follow as a confirmation.

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Clumping in both drops is an indication of auto agglutination, so a tube test

should be carried out. (TANKESHWAR ACHARYA, 2012)

Test tube

Uses rabbit plasma that has been inoculated with a staphylococcal The tube is

then incubated at 37 °C for 1hour 30 minutes. If positive the plasma will

coagulate, resulting in a clot (sometimes the liquid will completely solidify). If

negative, the plasma remains a liquid. The negative result may be S.epidermidis

but only a more detailed identification test can confirm this, using biochemical

tests A false negative can be resulted if the sample is not allowed to cool for

about 30 minutes at room temperature or 10 minutes in the freezer, the serum can

melt. If it’s a confirmed negative result, the serum will remain liquid after cooling.

(TANKESHWAR ACHARYA, 2012)

Picture 8 Picture 9

Picture 8: Slide tube. Coagulase positive shows clumping and coagulase negative

shows no clumping in either drop. (source: microbeonline.com)

Picture 9: Test tube. A fibrin clot is formed. (source: microbeonlie.com)

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Catalase Test

Spread the bacteria on an agar plate and inkubate the plate overnight (18-24

hours) under appropriate conditions. Collect bacteria from one colony with a

sterile inoculating loop (of plastic or platinum) and apply the bacteria on a

microscope slide. If the bacteria are collected from a blood agar plate, one has to

avoid contamination of agar, because hemoglobin also contains heme groups

which can cause a false positive reaction. Add one drop of 3% H2O2 to the

bacteria and observe the suspension. Positive test result is when oxygen gas forms

in the form av bubbles shows that the bacterium has a catalase. Negative test

result there is no gas formation. (TANKESHWAR ACHARYA, 2012)

D. Serological test.

Serological tests for antibodies to several different S.aureus antigens have been

used in diagnosing serious staphylococcal infections. These tests provide a

specific etiological diagnosis before blood cultures. Detection of elevated levels

of anti-staphylococcal antibodies supports a presumptive diagnosis of S. aureus

infection in patients with negative cultures because of prior antibiotic therapy. The

gel diffusion test is based on the precipitation produced by antigen/antibody

interaction, whereas the agglutination test is based on the agglutination of

antibody coated cells or particles. Double-gel diffusion procedures were reported

to assay SEA and SEB, with respective detection limits as low as 0.02 and 0.05

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µg per gram. A latex agglutination test used latex particles coated with specific

anti-SEB antibodies as indicators and the limit of detection was 0.2mL. (Wu.S,

2016)

E. Molecular diagnostic techniques

Involve isolating the DNA, RNA or protein of the prokaryotic cells and

identifying them based on sequence motifs. If in mannitol salt its fermented, the

acid produced turns the phenol red pH indicator from red (alkaline) to yellow

(acid). PCR has been widely used to detect SEs by amplifying corresponding

genes.The detection of SEs by PCR was first reported by Wilson et al, who used

two sets of primers to amplify the SEB and SEC genes and the staphylococcal

nuclease gene. Several PCR variants have been developed to detect SEs, such as

multiplex PCR, real-time PCR, reverse-transcriptase PCR, and loop-mediated

isothermal amplification (LAMP). Compared to the other PCR-based techniques,

the distinct advantage of multiplex PCR is simultaneous detection of several SEs

with different primers. PCR also has the advantage of being combined with other

techniques, such as most probable number (MPN-PCR), and PCR-enzyme linked

immunosorbent assay. PCR is much faster and can be applied to detect SEs in

most kinds of food, such as milk, cheese, and meat products. However, because of

interference with target-cell lysis necessary for nucleic acid extraction, nucleic

acid degradation and direct inhibition of PCR, false negative PCR results may

occur. (Wu.S, 2016)

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F. Diagnosis

Folliculitis, furuncle, and carbuncle

Diagnosis based on clinical appearance Aspiration or incision and culture of

purulent material from the lesion occasionally diagnosed. (Elizabeth P Baorto,

2016)

Osteomyelitis

Cultures of bone aspirate. Bone scan with increased technetium-99m–labeled

diphosphonate uptake supports the clinical diagnosis; however, this modality is

not as useful in neonates or after trauma or surgery. MRI is the best imaging

modality for defining purulent collections and for planning surgery. On plain film

radiographs, destructive bone changes are usually observed 2 weeks after

infection.

Septic arthritis

Gram stain and culture of joint fluid is the primary means of diagnosis. Direct

inoculation of synovial fluid into culture bottles may improve culture yield.

Median white blood cell count in joint fluid is 60.5 × 10 9, with neutrophil

predominance which is more than75%. Synovial fluid glucose levels are often

low. Plain radiographs show capsular swelling. (Elizabeth P Baorto, 2016)

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Endocarditis

Blood culture is the most important diagnostic procedure. Inject the blood sample

into hypertonic media if the patient has been exposed to antibiotics. Obtain 3-5

sets of large-volume blood cultures within the first 24 hours. Echocardiography is

a valuable adjunct. (Elizabeth P Baorto, 2016)

Pneumonia

Blood cultures are more likely to be positive in secondary than primary disease.

An adequate respiratory tract specimen should be obtained prior to initiating

therapy; specimens may include endotracheal sampling, pleural fluid, or lung tap.

Sputum specimens are inadequate because upper respiratory tract colonization is

common. No radiologic features are highly specific. Typical radiographic features

are unilateral consolidation in primary staphylococcal pneumonia and bilateral

infiltrates in secondary cases. Early in the disease course, the chest radiograph

may reveal minimal infiltrates, but within hours, infiltrates progress rapidly.

Pleural effusion, pneumatoceles, and pneumothorax are also common. In

oncology patients, S aureus may cause pulmonary nodules. (Elizabeth P Baorto,

2016).

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CHAPTER III

CONCLUSION

Staphylococcus aureus is gram-positive cocci that are microscopically observed

as individual organisms, in pairs, and in irregular clusters. is a round-shaped

bacterium and is frequently found in the nose, respiratory tract, and on the skin. S.

aureus infection spreads through contact with pus from an infected wound, skin-

to-skin contact with an infected person, and contact with objects used by infected

person.

The environmental factors that are found in staphylococcus aureus is from

infected wound and skin, marine environment and contaminated food.

There are many ways to identify Staphylococcus aureus. Cultured using blood

agar, mannitol salt agar, Baird Parker Agar (BPA). Biochemical test will be taken

by coagulate test which has two slide tube test and test tube test and catalase test.

Serological test for antibodies to several different S.aureus antigens have been

used in diagnosing serious staphylococcal infections. Bimolecular test such as

polymerase chain reaction is done. Several diagnosis such as folliculitis, furuncle,

carbuncle, osteomyelitis, septic arthritis, endocarditis, pneumonia and

thrombophlebitis.

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REFRENCE

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http://protomag.com/articles/a-brief-history-of-staph.

2. V. Chan, P. Sherman, B. Bourke, Bacterial genomes and infectious

diseases (Totowa, N.J. 2006).

3. Thaker, H., Brahmbhatt, M. and Nayak, J. (2013). Isolation and

identification of Staphylococcus aureus from milk and milk products and

their drug resistance patterns in Anand, Gujarat. Veterinary World, 5(12),

p.10.

4. Thaker, H., Brahmbhatt, M. and Nayak, J. (2013). Isolation and

identification of Staphylococcus aureus from milk and milk products and

their drug resistance patterns in Anand, Gujarat. Veterinary World, 5(12),

p.10.

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microscopy of Gram-positive cocci, morphology and microscopic

appearance of Staphylococcus aureus, S.aureus gram stain and colony

morphology on agar, clinical significanceAvailable at:

http://www.bacteriainphotos.com/bacteria%20under%20microscope/staph

ylococcus%20aureus%20microscopy.html (Accessed 10 Jul. 2017).

6. Sagar Ayal (2016). Mannitol Salt Agar for the isolation of Staphylococcus

aureus.pdf (Accessed 12 Jul. 2017).

7. Acharya, T. (2017). Coagulase Test: Principle, procedure and

interpretation - microbeonline. [online] microbeonline. Available at:

https://microbeonline.com/diagnostic-tests-biochemical-tests-coagulase-

test/ (Accessed 12 Jul. 2017).

8. Wu, S., Duan, N., Gu, H., Hao, L., Ye, H., Gong, W. and Wang, Z. (2016).

A Review of the Methods for Detection of Staphylococcus aureus

Enterotoxins. Toxins, 8(7), p.176.

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9. P Baorto, E. (2017). Staphylococcus Aureus Infection: Practice Essentials,

Background, Pathophysiology. Available at:

http://emedicine.medscape.com/article/971358-overview (Accessed 12

Jul. 2017).

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Picture 2 : Available at :

http://media.eol.org/content/2009/11/25/03/13786_580_360.jpg

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galore-microbial-pigments-and.html

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