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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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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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http://www.bacteriainphotos.com/bacteria%20under%20microscope/staph
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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
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https://microbeonline.com/diagnostic-tests-biochemical-tests-coagulase-
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A Review of the Methods for Detection of Staphylococcus aureus
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9. P Baorto, E. (2017). Staphylococcus Aureus Infection: Practice Essentials,
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