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School Sores 1. Common Bacterial Pathogens In this scenario, Stephanie is described to have red sores around her mouth and nose. The symptoms demonstrated align with impetigo (“Impetigo: Symptoms”, 2013), otherwise known as school sores, which is commonly caused by Streptococcus pyogenes or Staphylococcus aureus (Corfield, 2008). Since this situation occurs in an industrialized setting rather than tropical regions, it is more likely that S. aureus is the cause of impetigo (Bowen, Chatfield, & Carapetis, 2014) seen in Stephanie. However, co-infection with both bacteria has been observed, therefore after diagnosis, selecting treatment that is effective against both pathogens may be beneficial (Bowen, Chatfield, & Carapetis, 2014). S. pyogenes are Gram-positive bacteria that exhibit round-to-ovoid cocci that are both non-motile and non-sporulating (Patterson, 1996). They are considered to be Group A β-hemolytic Streptococci, commonly associated with respiratory as well as skin-related infections such as impetigo during childhood (Patterson, 1996). Similarly, S. aurerus are also Gram-positive cocci bacteria (Foster, 1996). Infection with S. aureus in the in the skin are commonly related to suppurative infections (Todar, n.d.), the formation of boils, furnucles, styes, and impetigo (Foster, 1996). 2. Samples and the Microbiology Laboratory Prior to taking samples of the sores, impetigo is usually diagnosed by physicians based on the appearance of the sores (“Impetigo: Tests and diagnosis”, 2013). Samples of the infected sites can be taken by swabbing the infected area, or using a blade to remove the crust of a pustule (Aly, 1996). In addition, samples of the pus can be taken and used for Gram staining (Aly, 1996) which can help in determining the bacteria present. After sample collection, it is ideal for swabs to be plated immediately. However, research has shown that dry swabs transported at 4oC with desiccant and plated within 48 hours, as wells as swabs inoculated into skim milk tryptone glucose glycerol broth, transported at 4oC, stored at -70oC and plated within 48 hours, are effective alternate methods to recover S. pyogenes and S. aureus from impetigo swabs (Bowen, Tong, Chatfield, Andrews, & Carapetis, 2013). The Microbiology laboratory is important in the diagnosis of this disease in order to determine whether impetigo was caused by S. pyogenes or S. aureus, or another pathogen since they may subsequently require different forms of treatments such as antibiotics or topical creams. Performing laboratory tests such as bacterial cultures can identify if there is antibiotic resistance present, as well as strain diversity, which will also influence treatment protocols. 3 & 4. Methods of Testing & Expected Results A) Culture and Gram Stain Analysis of the morphology of bacteria after growth in culture is a useful preliminary way to distinguish between S. pyogenes and S. aureus. As Group A streptococci, S. pyogenes preferentially grow at 37oC at 5-10% CO2, on complex growth mediums such as one with 1.4% Trypticase soy agar with 5% sheep blood (Gera & McIver, 2012). In particular, ideal growth media would be those that contain neo peptone extracts, glucose, and all amino acids (Gera & McIver, 2012). When viewed under a light microscope, we would expect to see S. pyogenes colonies in one plane direction in pairs, forming chains (Patterson, 1996). To identify S. aureus, the swab sample can also be streaked across blood agar or other solid culture media and then viewed under a light microscope after incubation at 37oC. The bacteria characteristically grow in two planes and form clusters (Foster, 1996) rather than the unidirectional plane chains exhibited by S. pyogenes. However since it is likely that the sample may contain other pathogens present, a culture method to isolate for S. pyogenes is to plate the sample on mannitol salt agar (as described in section B). When plated on blood agar, beta hemolysis is observed for both S. aureus and S. pyogenes since both bacteria exhibit haemolytic activity (Todar, n.d.). Beta hemolysis indicates complete lysis of red blood cells (RBC) and thus zones of hemolysis are observed around colonies in culture (Archaya, 2013). Other types of hemolysis include alpha hemolysis (partial lysis of RBC producing green or brown discoluration around a colony, seen in Streptococcus pneumoniae), and gamma hemolysis (no hemolysis of RBC) (Archaya, 2013). In particular, observing the type of hemolysis exhibited on blood agar cultures is useful in differentiating between different species of streptococcus. However, it is necessary to perform further tests such as bacitracin (section D) and CAMP (section E) tests to confirm the species. Similarly, in addition to hemolysis tests, to confirm the presence of S. aureus catalase tests (section C) and coagulase tests (section F) are reliable methods of identification. Performing a Gram stain of bacterial colonies is an efficient and inexpensive method to differentiate between groups of bacteria based on the color in which they stain, which is reflective of their cell wall composition (Su & Wang, 2011). During a Gram stain, cells are stained with crystal violet, then the dye is fixed with iodine treatment, then cells are de-colorized, and then counterstained with another dye such as safanin (Brucker, 1986). Gram-positive bacteria possess thick peptidoglycan in their cell walls, and therefore will stain violet when viewed under a microscope, while Gram-negative bacteria will stain red due to their thin peptidoglycan layers in their cell wall. Performing a Gram stain will confirm if there is in fact Gram-positive bacteria present (which can then lead to further tests to determine if it is the suspected pathogens S. aureus and S. pyogenes) or if there are Gram-negative bacteria present. Therefore, if S. pyogenes and S. aureus are present and a Gram stain is performed, it is expected to stain violet thus indicating Gram-positive bacteria are present. However, if Gram-positive bacteria are not present, the cells will stain red. B) Mannitol Salt Agar Plates Culturing the sample on mannitol salt agar (MSA) plates containing 7.5% sodium chloride is a useful way to isolate for, and thus identify the presence of Staphylococci (Foster, 2016). S. aureus are halotolerant and thus are able to grow and survive on MSA plates (Shields & Tsang, 2006). In addition MSA plates also reflect the bacteria’s ability to ferment mannitol through pH indicators whose colors reflect neutral conditions when red, acidic conditions when yellow, and basic conditions when pink (Shields & Trang 2006). Therefore, when cultured, it is expected that if S. aureus is present, the MSA plates will appear yellow as the bacteria are fermenting the mannitol sugar present. On the other hand, S. pyogenes are unable to, or have limited ability to, grow on MSA plates, nor are they able to ferment mannitol; therefore no color changes would be expected on MSA plates. C) Catalase Test A primary distinguishing characteristic between S. aureus and S. pyogenes is the presence of, or lack of enzyme catalase, respectively (Patterson, 1996). Therefore performing a catalase test would differentiate between the catalase-positive S. aureus and the catalase-negative S. pyogenes. Catalase enzymes are responsible for catalyzing the breakdown of hydrogen peroxide into water and oxygen gas (Reiner, 2010). During the test, samples of colonies are exposed to several drops of 3% hydrogen peroxide on microscope slides or in test tubes (Reiner, 2010). Therefore it is expected that catalase-positive bacteria cultures, such as S. aureus, will exhibit bubbles because the enzyme is releasing oxygen gas as it breaks down the hydrogen peroxide (Reiner, 2010). However, it is important that catalase tests are not performed on blood agar, because bubbles will also be observed due to the presence of red blood cells, thus creating a false-positive result (Patterson, 1996). On the other hand, it is expected that if the sample contains S. pyogenes, there would be no catalase activity, no bubbles observed and thus reported as a negative result for catalase test. D) Bacitracin Susceptibility Test A useful diagnostic method to determine the presence of S. pyogenes is to perform a bacitracin susceptibility test. As a Group A streptococci, the majority of S. pyogenes are sensitive to bacitracin antibiotic (Olender, Łetowska, Karyński, Kiernicka-Ciekot & Pels, 2011), therefore this test would be helpful in preliminarily determining if S. pyogenes are present in our sample, as well as differentiate S. pyogenes from other beta-hemolytic streptococci bacteria such as S. agalactiae which are resistant to the antibiotic. In the bacitracin susceptibility test, a bacitracin containing disc is placed in a blood agar culture and incubated at 37oC at 5% CO2 (Chamberlain, 2002). Since S. pyogenes are sensitive to bacitracin, it is expected that there will be a zone of inhibition around bacitracin-containing discs. On the other hand, other beta-hemolytic bacteria are expected to grow despite the presence of bacitracin, indicating that there may be other pathogens present in the sample. S. aureus possess two-component systems (TCSs) which are associated with susceptibility to antibiotics. TCSs in S. aureus sense bacitracin, and subsequently increase the activity of ABC transporters when antibacterial agents are sensed in the environment (Yoshida et al., 2011). This ultimately leads to increased bacitracin efflux and thus accounting for the decreased susceptibility of S. aureus to bacitracin (Yoshida et al., 2011). Therefore it is expected that minimal or no zones of inhibition will be observed when S. aureus are plated with bacitracin-containing discs. E) CAMP Test In combination with the bacitracin susceptibility test, CAMP tests can be performed to identify Group B beta-hemolytic Streptococci, such as S. agalactiae since some Group B streptococci can also be bacitracin sensitive. This method of testing is reliable as it rarely produces false-positive results with Streptococci (Archaya, 2013). S. pyogenes haemolytic activity is often enhanced by CAMP factor produced by Group B beta-hemolytic Streptococci. Therefore when S. pyogenes are plated on blood agar medium with Group B Streptococci, enhanced areas of hemolysis are expected. Since the swab sample is not expected to possess Group B Streptococci, it is expected that CAMP tests will be negative, and there will be a lack of enlarged areas of hemolysis. On the other hand, CAMP test results are expected to be positive if Group B Streptococci are present. It is also expected that CAMP test results will be negative for S. aureus since they do not produce CAMP factor. F) Coagulase Test Coagulases are polypeptides secreted by S. aureus that interact with prothrombin to convert the conversion of fibrinogen into fibrin, leading to clotting of plasma or blood (Cheng et al., 2010). Coagulase plays an important role in disease pathogenesis and persistence of S. aureus in host tissues therefore making it a useful marker for identification studies, allowing the differentiation between S. aureus and other strains of Staphylococci such as S. epidermidis, amongst other coagulase-negative bacteria. There are two different procedures for coagulase tests: slide test and tube test. In a slide test, a suspension of bacterial cells is mixed with a drop of EDTA-treated rabbit plasma on a microscope slide. Positive results (indicated by clumping) are indicative of the presence of bound coagulase, and thus S. aureus (Katz, 2010). In a tube coagulase test, a sample of bacterial cultures are mixed with a larger volume of EDTA- treated plasma and observed for clot formation within a 24 hour period of incubation at 37oC (Katz, 2010). Since S. aureus secrete coagulase, it is expected that clot formation will occur indicative of free coagulase, and thus test results will be reported as coagulase-positive. On the other hand, S. pyogenes are expected to be coagulase-negative since they do not secrete coagulase and thus are unable to form clots. The following table provides a summary of the expected results from each test discussed. Pathogen Test Streptococcus pyogenes Gram Stain Staphyloccocus aureus Expected Results Positive, violet stain Hemolysis Observed Colony Morphology Mannitol Salt Agar Plates Catalase Test Bacitracin Susceptibility Test CAMP Test Coagulase Test Gram Stain Beta Division in one plane, in chains Limited or no bacterial growth, no color change of MSA plate Negative - No bubbles Positive Hemolysis Observed Colony Morphology Mannitol Salt Agar Plates Catalase Test Bacitracin Susceptibility Test CAMP Test Coagulase Test Beta Division in two planes, clusters Bacterial growth, MSA plate color change to yellow Positive - Bubbles Negative Negative Negative Positive, violet stain Negative Positive Works Cited Aly R. Microbial Infections of Skin and Nails. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 98. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8301/ Archaya, T. (2013, August 22). Blood Agar: Composition, Preparation, Uses and Types of Hemolysis – microbeonline. Retrieved January 17, 2016, from http://microbeonline.com/blood-agar-composition-preparation-uses-and-types-ofhemolysis/ Archaya, T. (2013, November 8). CAMP Test: Principle, Procedure and results - microbeonline. Retrieved January 17, 2016, from http://microbeonline.com/camp-test-principleprocedure-results/ Bowen, A. C., Tong, S. Y., Chatfield, M. D., Andrews, R. M., & Carapetis, J. R. (2013). Comparison of three methods for the recovery of skin pathogens from impetigo swabs collected in a remote community of Northern Territory, Australia. Transactions of The Royal Society of Tropical Medicine and Hygiene, 107(6), 384-389. Bowen, A. C., Tong, S. Y., Chatfield, M. D., & Carapetis, J. R. (2014). The microbiology of impetigo in Indigenous children: associations between Streptococcus pyogenes, Staphylococcus aureus, scabies, and nasal carriage. BMC infectious diseases, 14(1), 727. Brucker, M. C. (1986). Gram staining A useful laboratory technique. Journal of NurseMidwifery, 31(3), 156-158. doi:10.1016/0091-2182(86)90154-0 Chamberlain, N. (2002). Growth Inhibition of Streptococcus pyogenes by Bacitraci. Available from: http://www.microbelibrary.org/library/laboratory-test/2963-growth-inhibition-ofstreptococcus-pyogenes-by-bacitracin Cheng, A. G., McAdow, M., Kim, H. K., Bae, T., Missiakas, D. M., & Schneewind, O. (2010). Contribution of coagulases towards Staphylococcus aureus disease and protective immunity. PLoS Pathog, 6(8), e1001036-e1001036. Corfield, J. (2008). Impetigo. In Y. Zhang (Ed.), Encyclopedia of global health. (pp. 888-889). Thousand Oaks, CA: SAGE Publications, Inc. doi: http://dx.doi.org/10.4135/9781412963855.n610 Foster T. Staphylococcus. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 12. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8448/ Gera, K., & McIver, K. S. (2012). Laboratory growth and maintenance of Streptococcus pyogenes (the Group A Streptococcus, GAS). Current protocols in microbiology, 9D-2. Hartman-Adams, H., Banvard, C., & Juckett, G. (2014). Impetigo: diagnosis and treatment. Am Fam Physician, 90(4), 229-235. Impetigo: Symptoms. (2013, May 15). Retrieved January 15, 2016, from http://www.mayoclinic.org/diseases-conditions/impetigo/basics/symptoms/con-20024185 Impetigo: Tests and diagnosis. (2013, May 15). Retrieved January 15, 2016, from http://www.mayoclinic.org/diseases-conditions/impetigo/basics/tests-diagnosis/con20024185 Katz, S. (2010, November 11). Coagulase Test Protocol. Retrieved January 17, 2016, from http://www.microbelibrary.org/library/laboratory test/3220-coagulase-test-protocol Olender, A., Łetowska, I., Karyński, M., Kiernicka-Ciekot, K., & Pels, K. (2011). Problems with identification of beta-hemolytic streptococcus resistant to bacitracin isolated from patients with pharyngitis. Medycyna doswiadczalna i mikrobiologia, 64(1), 1-10. Patterson MJ. Streptococcus. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 13. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7611/ Reiner, K. (2010). Catalase Test Protocol. Available from the MicrobeLibrary website: http://www.microbelibrary.org/library/laboratory+test/3226-catalase-test-protocol Shields, P., & Tsang, A. Y. (2006). Mannitol salt agar plates protocols. Available from the MicrobeLibrary website: http://www. microbelibrary. org/library/laboratory-test/3034mannitol-salt-agar-plates-protocols. Su, R. & Wang, P. (2011).Reviews in medical microbiology: Role of gram stain in microbiological laboratories with limited resources Lippincott Williams and Wilkins. doi:10.1097/MRM.0b013e3283478a08 Todar, K. (n.d.). Staphylococcus aureus. Retrieved January 15, 2016, from http://textbookofbacteriology.net/staph_2.html Yoshida, Y., Matsuo, M., Oogai, Y., Kato, F., Nakamura, N., Sugai, M., & Komatsuzawa, H. (2011). Bacitracin sensing and resistance in Staphylococcus aureus. FEMS microbiology letters, 320(1), 33-39.