The bacterial genus, Staphylococcus, will be isolated and identified in this project. This genus has been chosen to review because of its abundance on the skin of mammals and the pathogenic nature of one of its member, Staphylococcus aureus. Apart from skin infections, Staphylococcus aureus could mutate to form Methicillin-resistant Staphylococcus aureus (MRSA), which shows resistance to antibiotics. In both cases, these give rise to medical implications. In addition, the distinctive features of Staphylococcus aureus have increased the ease to isolate and identify it from other species in the genus via culturing and biochemical tests.
The aim of the project is to isolate Staphylococcus aureus from a bundle of cat hairs and verify its identity via microscopic examination and biochemical tests. No human specimen is used due to the potential pathogenic property of the bacterium. It is intended that a pure culture of pathogenic Staphylococcus aureus is obtained. For the purposes of this project, the importance of Staphylococcus aureus to humans, its classification in terms of morphology and physiological properties, methods of isolation with the use of growth media and the technique of streak plating and identification by biochemical tests would be the four objectives to be addressed.
Objective 1: Importance of Staphylococcus aureus to humans The importance of Staphylococcus aureus to humans would be outlined by a review of its cell structure, cell physiology and environmental niches, followed by the medical implications of Staphylococcus as a result of these properties.
As a member of the Bacteria domain, it is expected that Staphylococcus has bacterial cell structure. In other words, it lacks nucleus and membrane-bound organelles. The structural elements in a cell of Staphylococcus should include a cell membrane, cell wall, ribosome and nucleoid (6).
Moreover, being one of the five genera from the family of Staphylococcaceae, Staphylococcus possesses specific cellular properties that are unique to this family. In particular, it is a cocci and gram-positive bacterium and this indicates that its cell wall is essentially composed of a thick layer of peptidoglycan (21).
In addition to the above structures, Staphylococcus aureus possesses some special cellular structures that distinguish it from other species in the genus. This includes the possession of surface proteins that help attachment to proteins such as the fibronectin and fibrinogen-binding proteins involved in blood clotting (3). This cellular property may explain the pathogenic nature of Staphylococcus aureus, as infections might be caused by invasion via wounds.
On the other hand, it is worthwhile to note that Staphylococcus does not have flagella and spores (16). That is to say, Staphylococcus aureus is non-motile.
The cell physiology of Staphylococcus covers temperature, pH and oxygen requirements.
Most Staphylococcus can grow at 45°C, but it is reasonable to predict that its optimal temperature for metabolism would be close to the body temperature of humans, which is 37°C (5).
Concerning the optimum pH for metabolism, the enzymes in Staphylococcus work best in slightly alkaline medium, with a pH range of 7.4 to 7.6 (16).
As for oxygen requirement, Staphylococcus is facultative anaerobic (21). This implies that Staphylococcus can grow regardless of the presence of oxygen, but the presence of oxygen would be more favorable.
In the presence of oxygen, Staphylococcus utilizes glucose to carry out cellular respiration to generate energy for metabolism. Oxygen performs the role of a terminal electron acceptor and it is completely reduced to water (8).
When oxygen is lacking or absent, Staphylococcus may undergo fermentation and lactic acid is the usual product (21). In the process, glucose is converted into substrate pyruvate, followed by its binding to the cofactor Nicotinamide Adenine Dinucleotide (NAD ) to produce lactic acid (6).
Moving on the ways Staphylococcus metabolize, as light is not readily available on skin surface and mucous membranes, it is proposed that Staphylococcus obtain energy via organic chemical compounds. Hence it is regarded as a chemotroph (21). The facultative anaerobic property of Staphylococcus may lead to a deduction that it utilizes organic carbon as the source of electron when oxygen is present. Though some Staphylococcus may use reduced forms of inorganic nitrates to generate electrons, its preference towards an aerobic atmosphere should define it as an organotroph (21). When comes to carbon source, Staphylococcus is a heterotrophy (12). That is to say, it attains its carbon source by utilization of organic substances such as sucrose for synthesis of metabolites (19). To summarize, Staphylococcus should be one of the members of the microbial group, Chemo-organotrophic heterotrophs.
The environmental niches of Staphylococcus can be addressed by its interactions with the environment as to where it is found, the type of relationship it forms with other organisms and its capability of undergoing mutation.
Staphylococcus is commonly found on the skin and mucous membranes of animals with stable body temperatures, including humans (15). Typically, the skin temperature of humans is approximately 32°C, which is reasonably close to the optimal temperature of 37oC (22). This enhances the growth of this microbe on skin. Moreover, the salty environment along skin surface due to the production of sweat may also account for the abundance of Staphylococcus in humans, since its enzymatic activity is optimal at more alkaline pH (17).
Staphylococcus aureus specifically colonizes in nasal cavity, larynx and on the skin surface of humans (2). The colonization of Staphylococcus aureus is principally achieved by fibrinogen-binding proteins adhering to the epithelial cells of the humans and thus this may outline a host-parasitic relationship between Staphylococcus and humans (10).
The interactions of Staphylococcus with the environment may also be underlined by mutation, which often occurs with Staphylococcus aureus. An example would be Methicillin-resistant Staphylococcus aureus (MRSA), a Staphylococcus aureus that is resistant particularly to the antibiotic, Methicillin (21). The mutation is caused by an alteration of the methicillin-resistance gene (mec A) coding for a penicillin-binding protein (4). This results in failure of antibiotics to cure infections caused by Staphylococcus aureus, which will be addressed in the medical implication section.
Medical implications of Staphylococcus
The features as in the cell structure, cell physiology and environmental niches of Staphylococcus can pose a great diversity of medical implications, which presents the importance of this bacterial genus.
Statistics show that Staphylococcus aureus is present in 79% of healthy people (14). Though Staphylococcus may colonize on the skin surface of the host without causing any harms, its ubiquity can still present various medical issues. The MRSA mentioned previously would be one of the problems associated with Staphylococcus. Apart from methicillin, MRSA could show resistance against many other antibiotics such as penicillin and amoxicillin (1). The ineffectiveness of existing antibiotics to cure MRSA infections has resulted in fatality, and it is usually characterized by the incidence of septic shock and pneumonia (11). A rapid increase of MRSA infections has been observed over the decades. The rate of hospitalized MRSA infections was only 2% in 1974 but this figure increases dramatically to approximately 40% in 1997 (13). Consequently, this causes deaths of 19000 in the United States of America annually (11).
As Staphylococcus colonies on skin surfaces and mucous membrane, skin infections and diseases associated with mucous membranes could be another medical implication. It is known that Staphylococcus aureus may cause Scalded Skin and Toxic Shock syndromes. Moreover, it may cause urinary tract infections and food poisoning (9).
Objective 2: Classification of Staphylococcus The classification of Staphylococcus can be reviewed in terms of its morphology and some of the physiological properties stated above.
The morphology of Staphylococcus can be described as cocci gram positive bacteria arranged in a cluster, which can be readily observed under microscope with the application of gram stain. A purple color would be observed.
The reason for its cluster formation may be explained by its capability of undergoing binary fission in multiple planes with daughter cells remains proximal to each other (16).
In terms of thermal requirement, Staphylococcus is classified as a mesophile. Regarding pH requirements, it falls into the category of neutrophile. Moreover, being a facultative anaerobe, Staphylococcus is catalase positive and it is generally considered a chemoorganotrophic heterotroph. In addition, Staphylococcus aureus is coagulase positive but not for other species in the genus. The absence of flagella indicates that Staphylococcus is a non-motile bacterium.
Objective 3: Methods of Isolation of Staphylococcus The methods of isolation of Staphylococcus would include growing in medium followed by streak plating.
To ensure optimum growth of Staphylococcus colonies, the sample of cat hairs should be enriched in nutrient broth with sodium chloride (NaCl) before plating on a nutrient agar. A nutrient broth normally consists of beef extract and peptone as fuels for growth (21). The temperature of incubation should be 37°C and the duration of incubation should be at least a day (20). This ensures that the Staphylococcus isolated can have sufficient time to grow at its optimum temperature. The addition of salt allows for a selective medium for Staphylococcus as it predominantly grows in salty environment. It also increases the pH of the medium to provide for a more alkaline environment to facilitate growth.
Alternatively, a growth medium can be done via a Mannitol salt agar (MSA), which consists of 7.5% of NaCl and phenol red as a pH indicator. The medium is then incubated at 37°C for two days (14). MSA essentially acts as both a selective and differential medium. NaCl selects for saline-favored Staphylococcus and the pH indicator differentiates between Staphylococcus aureus and Staphylococcus epidermidis. Differentiation can be illustrated by the fact that Staphylococcus aureus utilizes mannitol in the agar for metabolism, and the generation of acidic product is indicated by a yellow color. However, this phenomenon does not apply to Staphylococcus epidermidis (21).
Following enrichment, Staphylococcus in the medium can be transferred to an agar plate with nutrient broth and salt, by employment of aseptic techniques. At the same time, a transfer to an agar plate with only nutrient broth should be performed as a control set-up. This is to ensure the effectiveness of the selective media because other bacteria could grow on the agar plate if the medium was not set up properly.
Afterwards, the plates would be incubated for a week at 37oC for at least a day as in the incubation of sample in the nutrient broth. Plating and incubation should be repeated a few times to make sure that the colonies grown are pure.
Objective 4: Identification by biochemical tests The identity of Staphylococcus cannot be confirmed by carrying out the gram reaction alone due to the fact that a great variety of bacteria from other genus may also show gram positive reaction. Therefore, some biochemical tests have to be performed to verify that the bacteria isolated is in the genus of Staphylococcus and it is of the species Staphylococcus aureus. The catalase, Hugh and Leifson’s oxidation fermentation and cogulase tests are regarded as the standard tests for identification of Staphylococcus aureus (18). The mechanism of the tests is outlined below.
First of all, as Staphylococcus aureus is facultative anaerobic, it is expected that it contains enzymes to break down harmful products generated along the pathways of aerobic respiration. For instances, catalase breaks down superoxide radical hydrogen peroxide (H2O2) to oxygen and water, which are less harmful (8). Therefore colorless gas bubbles can be observed when H2O2 is added to a colony of Staphylococcus aureus.
Moreover, this property allows the Hugh and Leifson’s oxidation fermentation test to be performed. The bacterial sample is inoculated in a tube of Hugh
Case Study: Liver Disease and Hepatitis B
Case study – Liver Disease Introduction
A 60 year old woman with a history of hepatitis B and cirrhosis presents with oedema and constipation. The GP takes the following tests on her and these are the results.
Platelet count <1×10^11 platelets per litre.(portal hypertension)
Alpha – fetoprotein 450 ng/mL
The test results can be used for analysis to clarify what disease the 60 year old woman has, and by linking her symptoms with the test results. Hepatitis B is a virus that affects the liver, chronic hepatitis can develop without proper treatment. Having hepatitis B will eventually cause scarring to the liver which is known as cirrhosis. The 60 year old woman has developed Chronic hepatitis, the hepatitis is ongoing and serious, this will eventually cause the liver tissue to produce scares and stop functioning as it should .A liver biopsy (tissue sample) can be taken to find out how serious the hepatitis is. One of the function of the liver is to produce a protein called albumin, having liver cirrhosis will cause permanent damage to the liver and when this happens the liver will find it had to produce albumin.(Aspinall et al 2011:Pubmed) Albumin is a protein that is made by the liver which measures the exact amount of protein in the clear liquid portion in blood. The test is usually taken place to determine whether a person has a sort of liver or kidney disease which is the main reason why the test was carried out. The normal range in human is between 3.4 – 5.4g/dL. The woman’s albumin level shows that it is below average, low albumin may cause oedema which is a symptom that the woman is experiencing. Cirrhosis results in an increase in fluid retention. Cirrhosis also leads to low levels of albumin and other proteins in the blood which could also be the cause of oedema. (Gupta and Lis, 2010)
Platelet count determines the amount of platelet in the blood, normal adults produce 1 x10^11 platelets every day, portal hypertension is a complication of cirrhosis of the liver. Portal hypertension always takes place in the liver at all times, an increase in the pressure within the portal vein is caused by barrier of the blood flow to through the liver (Kotoh et al 2012). Portal hypertension causes symptoms to patients that are linked to their liver disease which could be hepatitis B,C or cirrhosis, patients that have cirrhosis had a very high chance of developing portal hypertension and it was increase over years. Patients who have portal hypertension usually have low platelet count which is the case of the 60 year old woman whose platelet count is <1011 /L which is below average. Thrombocytopenia refers to low blood platelet count. Thrombocytopenia has many causes, including cancer, medications, disease and infections. Thrombocytopenia is a common indicator of cancer, because sometimes the cancer and drugs used to treat the cancer affect platelet production in the bone marrow. (Kotoh et al 2012).
Alpha fetoprotein is a protein that is found in the liver, it is considered a tumour marker for liver cancer, the test may be done to diagnose possible liver diseases. The normal values in males or non-pregnant females is generally less than 40 micrograms/litre.High levels of alpha-fetoprotein indicates that there could be liver cancer which is the key factor which shows that there is something wrong with the liver.If your AFP level is unusually high but you are not pregnant, it may indicate the presence of certaincancers or liverconditions.so a liver transplant could be an option for the woman. In adults, high blood levels can be a sign of certain types of cancer, including liver cancer. (Alejandro et al, 2012)
Symptoms and Diagnosis
Constipation is a liver related issue, in this case It was most likely caused by her progressing tumour formation this may press on her digestive system causing constipation, having cirrhosis may also not allow her to properly digest fatty lipids because her bile might possibly be blocked resulting in constipation. Hypothyroidism can cause constipation. Blood tests to check thyroid hormones / TSH will help in diagnosing hypothyroidism. Oedema is swelling that is caused by fluid trapped in the bodies tissues, oedema can be a sign of lots of things including malnourishment and low albumin level in the blood and the woman’s albumin level is below average these are the results of having liver failure and cirrhosis. Having history of cirrhosis is the main reason she has oedema. (Gaw et al 2013)
Because of the woman’s age cancer would need to be tested for cancer as old people are much more prone to have cancer. Albumin is produced by the liver meaning that her liver is affected inducting liver cancer. Some patients with chronic liver diseases are more likely to develop thyroiditis, hyperthyroidism or hypothyroidism through autoimmune mechanisms (Huang, Liaw 2008). The woman may have Hepatocellular Carcinoma (HCC) which is most common and popular type of liver cancer. The main risk factors associated with HCC are hepatitis B,C and cirrhosis which the woman has a history of. HCC develops in patients with chronic liver disease and patients with cirrhosis are more likely to develop HCC and people over the age of 50 are more likely to develop HCC. The most common diseases that affects the liver are cirrhosis and hepatitis (Marshall and Bangert 2008). HCC is a serious disease in which alpha-fetoprotein will be elevated in a person, alpha-fetoprotein is a gene that becomes expressed when lots of damage has occurred to the liver in HCC. The normal range for AFP is 10-20 ng/mL.A level of >400 ng/mL may be regarded as diagnostic for HCC by some.
A test that can be proposed is an ultrasound of the liver, an ultrasound test uses sound waves to create pictures and to see what is going on with organs inside the body. If any tumours are found in the liver then this can be furthered to test for cancer. An MRI scan can be useful for looking at liver cancers, MRI are sometimes good at telling which cancers are a tumour. Another tests that can be carried out is biopsy which involves actually taking the tumour and inspecting it under the microscope for further analysis which is likely to be more effective and reliable. Alanine aminotransferase(ALT)An alanine aminotransferase (ALT) test measures the amount of this enzyme in the blood. ALT is found mainly in the liver, ALT is measured to see if the liver is damaged or diseased. Low levels of ALT are normally found in the blood. But when the liver is damaged or diseased, it releases ALT into the bloodstream, which makes ALT levels go up. Most increases in ALT levels are caused by liver damage.
Treatment and Prognosis
Some treatment for HCC can include a liver transplant or surgery which can remove small or small-growing tumours.Sorafenib tosylate (Nexavar), which is an oral medication can be taken to block and stops tumour from growing.The prognosis is often poor, because only 10 – 20% of hepatocellular carcinomas can be removed completely using surgery. Radio frequency ablation can be used to kill cancerous cells. If the cancer cannot successfully be removed or killed, the disease is usually deadly within 3 – 6 months. However, this is not always the case as everybody is difference and so on some occasions people will survive much longer than 6 months. (Forner at al 2012: Pubmed)
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Allan Gaw, Michael J. Murphy, Rajeev Srivastava, Robert A. Cowan, Denis St. J. O’Reilly. (2013). Clinical Biochemistry. 5th ed. London: Churchill Livingstone. p197-202.
Aspinall EJ, Hawkins G, Fraser A, Hutchinson SJ, Goldberg D.. (2011). Pubmed: Occupational Medicine. 8 (2), p531-540.
Digant Gupta and Christopher G Lis. (2010). Nutrition Journal. Pubmed: Pretreatment serum albumin as a predictor of cancer survival: A systematic review of the epidemiological literature. 9 (69), p112-120.
Feldman M, Friedman LS, Brandt LJ, Pratt DS (2010). Liver chemistry and function tests. 9th ed. Philadelphia: Saunders Elsevie. p436-450.
Kazuhiro Kotoh, Marie Fukushima, Yuki Horikawa, Shinsaku Yamashita, Motoyuki Kohjima, Makoto Nakamuta, Munechika EnjojiJanuary. (2012). Experimental and theurapitcal mediine. Serum albumin is present at higher levels in alcoholic liver cirrhosis as compared to HCV-related cirrhosis. 3 (1), p166-170.
Miau-Ju Huang and Yun-Fan Liaw. (2008). Wiley Online Library. Journal of Gastroenterology and Hepatology. 10 (3), P237–364.
William J Marshall, Stephen K Bangert (2008). Clinical chemistry. 6th ed. Edinburgh: Mosby Elsevier. p40-50.