FRANCHESKA CAMILO


Autor: Francheska Camilo González
November 10, 2013
English version: February 1, 2014


Laboratory Exercise for Medical Microbiology: Bacterial Identification of Gram – Positive Cocci


ABSTRACT

 
Pathogenic bacteria such as Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae belong to the group of gram-positive cocci, causing disease and nosocomial infection that is increasingly affecting healthy individuals in the community. This has led to the clinical and microbiological testing laboratory to be used consecutively to find treatments for resistant bacterial strains and to identify the bacteria causing a specific disease and thus give the antibiotic or treatment. Through this lab exercise is conducted the catalase test, Gram stain, hemolysis, and susceptibility test to bacitracin and optochin in pathogenic bacteria (Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes). It was determined that the Staphylococcus aureus is a catalase positive, Streptococcus pneumoniae is susceptible to Optochin, and Streptococcus pyogenes is susceptible to bacitracin. It also was found that Staphylococcus aureus and Streptococcus pyogenes are beta hemolysis, whereas, Streptococcus pneumoniae is alpha hemolytic and that the three bacteria are gram-positive.
 
Keywords: Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Microbiology Laboratory, laboratory tests, the gram-positive cocci

 


CONTENT
 
Prepared by: Francheska Camilo González
November 10, 2013
English version: February 1, 2014
ABSTRACT
ILLUSTRATIONS
INTRODUCTION
STAPHYLOCOCCUS
STAPHYLOCOCCUS AUREUS
Figure 1: Staphylococcus aureus
Figure 2: Cutaneous abscess located on the back and caused
by Methicillin-resistant Staphylococcus aureus bacteria (MRSA)
STREPTOCOCCUS
Figure 3: Streptococcus pneumoniae
STREPTOCOCCUS PYOGENES
Figure 4: Pharyngitis by Streptococcus pyogenes
STREPTOCOCCUS PNEUMONIAE
Figure 5: Pneumoonia-infected right lung by Streptococcus pneumoniae
Table 1: Diseases and infections by Staphylococcus aureus, Streptococcus pyogenes,
and Streptococcus pneumoniae
Part 2: Practice of Medical Microbiology Laboratory to identify gram-positive cocci
Laboratory group: Kelvin Garced  , José Noy , Francheska Camilo
BACKGROUND
MATERIALS / METHODS
Figure 6: Hemolysis Beta (β) for the bacteria Streptococcus pyogenes
and Staphylococcus aureus in blood agar plate (Laboratory exercise: plate 3)
RESULTS
CONCLUSION
REFERENCES



ILLUSTRATIONS
 
Figure 1:   Staphylococcus aureus
Figure 2: Cutaneous abscess located on the back and caused by Methicillin-resistant Staphylococcus aureus bacteria (MRSA)
Figure 3:   Streptococcus pneumoniae
Figure 4:   Pharyngitis by Streptococcus pyogenes
Figure 5:   Pneumoonia-infected right lung by Streptococcus pneumoniae
Figure 6: Hemolysis Beta (β) for the bacteria Streptococcus pyogenes and Staphylococcus aureus in blood agar plate (Laboratory exercise: plate 3)




INTRODUCTION
 
The presence of microorganisms such as viruses, fungi, parasites, and pathogenic bacteria, causes the use of microbiological techniques is needed to identify these infectious agents in the urine, feces, blood, sputum, and in other body fluids, in persons with compromised immune systems or diseases related to these microorganisms. Microbiologists consistently perform immunological tests and other experiments chemical, genetic and microbiological, where methods and research techniques are used to identify pathogens. The sputum culture, stool test, urine culture, sensitivity of burns and wounds, and blood cultures are examples of microbiological tests. Microbiologists also use microbiological tests such as Gram stain, catalase test , and test sensitivity to optochin and bacitracin, among other experimental methods of analysis to identify bacteria in a group composed of different species, as an example, our laboratory exercise, where we made an analysis using these experimental tests to identify bacteria Cocci Gram-positive (Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus), which cause diseases and serious infections [3] .




Part 1: What are the Gram-positive Cocci?

Gram - positive cocci are spherical bacterial cells. These bacteria are characterized by reaction to Gram staining, and the absence of endospores [1]. In this group of bacteria was analyzed Staphylococcus aureus species of genus Staphylococcus, and species Streptococcus pneumoniae and Streptococcus pyogenes, of genus Streptococcus.



STAPHYLOCOCCUS

 
Staphylococcus, also known as staphylococci, is a bacterial genus of gram - positive cocci that is located in the normal flora in the skin and mucosal surfaces. These bacteria can be classified as a pathogenic group that can cause infections in the skin, soft tissues, bone and genitourinary tract. These cells have a pattern like a bunch of grapes, but in samples at the clinical level, these microorganisms can occur as single cells, in pairs, or short chains. Most of these bacteria are facultative anaerobic (can increase in anaerobic or aerobic environments), and are able to grow in media with elevated concentrations in salt, and in temperatures of 18- 40°C [1].




STAPHYLOCOCCUS AUREUS
 
Staphylococcus aureus is a bacterium that is gram-positive, catalase-positive, and is characterized by presence of a thick layer of peptidoglycan. These bacteria without outer membrane can be found in the normal flora of the skin, mucosal surfaces, and spread is from contact between people or with contaminated surfaces. Staphylococcus aureus can survive in dry areas for long periods of time. The virulence factors of this bacterium are the structural components (Capsule, Layer of extracellular polysaccharides, peptidoglycan, teichoic acid, protein A), toxins (Cytotoxins, exfoliative toxins (ETA, ETB), enterotoxins, Toxin 1 TSS), and enzymes (Coagulase, Hyaluronidase, Fibrinolysin, lipases, and nucleases). Risk factors are the presence of foreign bodies (prosthesis, catheter), previous surgery, and the use of antibiotics that inhibit the normal microbial flora necessary in the human body. MRSA (Staphylococcus aureus resistant to methicillin) is a bacterium that causes severe nosocomial infections, and extra-hospital infections in children and adults previously healthy [1].

Antibiotics that should be used to Staphylococcus aureus are:[1]

 
  • Oxacillin (or other penicillinase-resistant penicillins)
  • Vancomycin (for strains that are resistant to oxacillin)
  • Trimethoprim-sulfamethoxazole, clindamycin, linezolid, quinupristin-dalfopristin or daptomycin (All antibiotics mentioned are alternatives that may be used as antibiotics to treat infections caused by MRSA).

Figure 1: Staphylococcus aureus

Source: Samaritan Infectious Disease page.
<
http://www.samaritanid.com/staphylococcus_aureus.html> Accessed: 11/8/13



Staphylococcus aureus Resistant to Methicillin (MRSA) is a major problem worldwide, as this has become one of the causes of skin infection, and in more recent cases, is the cause of invasive infections in children and healthy adults in the community[1, 4]. In Research Publication, "Vancomycin versus linezolid in the treatment of methicillin -resistant Staphylococcus aureus meningitis in an experimental rabbit model", is compared the antibacterial efficacy of treatments such as linezolid and vancomycin in an experimental rabbit model (MRSA) meningitis. In this study, meningitis was induced by intracisternal inoculation using the ATCC 43300 strain. After 16 hours of incubation and development of meningitis, the control group received no antibiotic, the experimental group  "vancomycin" received (vancomycin 20mg/kg) every 12 hours, the experimental group "linezolid 10" received (linezolid 10mg / kg) every 12 hours and the experimental group "linezolid 20" received (linezolid 20mg/kg) every 12 hours. He proceeded to count bacteria from cerebrospinal fluid to 16 hours of incubation and 24 hours after treatment, and found that at 16 hours, the bacterial count was similar in all experimental groups, but at the end of treatment (24 hours) in the vancomycin group the value obtained was 2 logs higher than obtained for the linezolid group 20 (p> 0.05), and 4 logs higher than obtained for the linezolid group 10 (p=0.037). Obtaining the full or partial bacteriological response was greater in vancomycin vs. "Linezolid-10", but not in the comparison of the results obtained for vancomycin versus "linezolid-20". Therefore, by this research is suggests that treatment "linezolid" is not statistically inferior to treatment with vancomycin, when these were tested in the treatment (MRSA) meningitis, using an experimental model of rabbit at a dose of linezolid 20 mg / kg, but the treatment was less for a dose of linezolid when being used 10 mg / kg[5] .


Figure 2: Cutaneous abscess located on the back and caused by Methicillin-resistant Staphylococcus aureus bacteria (MRSA).

Photo credit: Gregory Moran, M.D.
Source: Centers for Disease Control and Prevention page.

<http://www.cdc.gov/mrsa/community/photos/photo-mrsa-10.html>
Accessed: 11/8/13.



STREPTOCOCCUS

The Streptococcus is bacteria that are gram-positive cocci, which are arranged in pairs or chains. Most are facultative anaerobes, but some only grow in an atmosphere enriched with carbon dioxide (CO2). Streptococci are bacteria that for be isolated need an enriched blood or serum, and they can ferment carbohydrates and generate lactic acid by this process. The species of streptococci are catalase-negative, and these stand out as pathogenic bacteria to humans. Species of this genus are classified by the following three systems: 1) the serological properties: Lancefield groups (A - W); 2) hemolytic patterns: complete hemolysis (beta [β]), incomplete hemolysis (alpha [α]) and absence of hemolysis (gamma [γ]); and 3) the biochemical properties (physiological)[2] .



Figure 3: Streptococcus pneumoniae

Source: International Society of Microbial Resistance page.
<
http://www.microresistance.org/amdr.cfm?CEProgramID=95&HeaderCEProgramContentID=85>
Accessed: 11/8/13.



STREPTOCOCCUS PYOGENES

Streptococcus pyogenes is a species of the genus Streptococcus. These bacteria are gram-positive cocci that are arranged in chains and have rapid growth. In the interior of the cell wall of this bacteria is located the antigen group A of Lancefield and the protein M, which is associated with the virulent streptococci. Streptococcus pyogenes is spread by the contact between people for respiratory droplets (pharyngitis) and the direct contact with an infected person in a skin rupture. This bacterium is identified because is positive for PYR and susceptible to bacitracin, whereby the general treatment used for patients with this bacterium is penicillin[2] .

People most at risk of developing a disease by Streptococcus pyogenes are:[2]

 
  • Children 5 to 15 years (pharyngitis)
  • Children between 2-5 years with poor hygiene (pyoderma)
  • Patients with soft tissue infection (streptococcal toxic shock syndrome)
  • Patients with a history of pharyngitis (rheumatic fever, glomerulonephritis)

Figure 4: Pharyngitis by Streptococcus pyogenes

Source: Grupo de Infecciosas SoMaMFYC page.
<http://grupoinfeccsomamfyc.wordpress.com/category/microorganismos/bacterias/streptococcus-pyogenes>
Accessed: 11/8/13.



STREPTOCOCCUS PNEUMONIAE

Streptococcus pneumoniae is a species of the genus Streptococcus, which is formed by gram-positive cocci, which have the form of "lancet" and are arranged in pairs (diplococci), or short chains. These bacteria have teichoic acid rich in polysaccharide C (phosphocholine) and an autolytic enzyme in the cell wall. The virulence of Streptococcus pneumoniae is because these bacteria can colonize the oropharynx, and extend to sterile tissue, being capable of inciting the local inflammatory response (peptidoglycan fragments, teichoic acid, pneumolysin) and escape the phagocytosis. Streptococcus pneumoniae infections are transmitted from person to person and are caused by the endogenous spread from the nasopharynx to distant regions (lungs, ears, blood, meninges and sinuses). Colonization is highest in young children, and diseases caused by this organism are more common in the colder months, because people stay home and tend to have more contact in confined spaces. This bacterial strain can be identified because is detectable in laboratory test like catalase-negative, susceptible to optochin and soluble in bile. The general treatment is penicillin and immunization methods (conjugate vaccine of 7 serotypes for children of 2 years of age, and the polysaccharide vaccine of 23 serotypes for adults at risk of acquire the disease caused by Streptococcus pneumoniae)[2, 7].


Figure 5: Pneumoonia-infected right lung by Streptococcus pneumoniae

Source: University of Michigan page. <http://sitemaker.umich.edu/mc2/pneumonia> Accessed: 11/10/13.



Table 1: Diseases and infections by Staphylococcus aueus, Streptococcus pyogenes, and Streptococcus pneumoniae
[1, 2]






Part 2: Practice of Medical Microbiology Laboratory to identify gram-positive cocci

Laboratory Group: Kevin Garced [K-G], Jose Noy [J-N], Francheska Camilo [F-C]


Method and laboratory exercise: Livier González [L-G], William Arias [W-A]

[L-G] Medical Microbiology’s Professor at the Interamerican University of Puerto Rico.
[W-A] Microbiology’s Professor at the Interamerican University of Puerto Rico.
[K-G, J-N, F-C] Students of the InterAmerican University of Puerto Rico.



BACKGROUND

The aim of this study was to identify bacteria gram-positive cocci in microbiology's tests (Gram staining, catalase test, and susceptibility to bacitracin and optochin).


MATERIALS / METHODS [L-G, W-A]

For the analysis, the procedure of Practical exercise was used: Streptococcus by Dr. Livier González [L-G]. The bacteria used in the experiment were: Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae. For the lab exercise, we proceeded to divide four blood agar plates in the center (with a Sharpie) to prepare individual crops of gram-positive cocci bacteria. For the experimental practice, we conducted a simple striated for the boards (3A, 4A), and a confluent striated for the blood agar plates (3B, 4B), to do the susceptibility test to bacitracin and optochin. The blood agar plates were inoculated as follows: the plate 3A (Streptococcus pyogenes and Staphylococcus aureus), the plate 4A (Streptococcus pneumoniae and Staphylococcus aureus), the plate 3B (Streptococcus pyogenes and Staphylococcus aureus) and the plate 4B (Streptococcus pneumoniae and Staphylococcus aureus).

We proceeded to add a bacitracin disk for each bacteria in plaque 3B, and an optochin disk each bacteria in plaque 4B. Then we proceeded to incubate the four plates at 37 ° C using a jar with CO2 for a period of 24 hours. For this exercise laboratory, we also conducted a test to determine the production of the enzyme catalase. For this, we proceeded to prepare three microscope slide laminae with one or two drops of 3% peroxide and a colony of one bacteria (Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae) for each slide, and thus to observe and analyze the production of bubbles (O2). In the next lab period (after incubating the blood agar plates for a period of time> 24 hours), we prepared the Gram stain with each bacterium using the procedure Laboratory # 5: Gram stain, by Dr. William Arias [W-A]. The procedures for the Gram stain were carried out as follows: prepare smears, drying this at room temperature, fix with heat, cover with crystal violet dye for 1 minute, rinse with water for 30 seconds, cover with iodine solution (mordant) for 1 minute, rinse with water for 30 seconds, discolor with alcohol (95% ethanol) for 15-30 seconds, rinse with water for 30 seconds, dye with safranin (counterstain) for 1 minute, rinse with water for 30 seconds, dry with bibulous paper (blot dry), and then we observed each slide prepared in the microscope using the oil immersion [6]. After completing our laboratory exercise, we proceeded to collect and analyze the results for the catalase test, Gram stain, hemolysis and susceptibility testing using bacitracin and optochin.



Figure 6: Hemolysis Beta (β) for the bacteria Streptococcus pyogenes and Staphylococcus aureus in blood agar plate (Laboratory exercise: plate 3)
Photo credit: Kelvin Garced [K-G] , Jose Noy [J-N],  Francheska Camilo [F-C]
Source: Medical Microbiology Laboratory's Exercise for identify bacteria coccus gram positive.
www.FrancheskaCamilo.com


RESULTS

In the exercise of Laboratory of Medical Microbiology "identification of gram-positive cocci bacteria", was performed, the catalase test, hemolysis, Gram stain and susceptibility using optochin and bacitracin in samples prepared with the bacteria Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes. In the catalase test, it was found that Staphylococcus aureus bacteria are catalase-positive, whereas Streptococcus pneumoniae and Streptococcus pyogenes are catalase - negative. For the susceptibility test to optochin and bacitracin, we find that the Streptococcus pneumoniae is susceptible to optochin, and Streptococcus pyogenes is susceptible to bacitracin. In this laboratory exercise, was obtained that the Streptococcus pyogenes and Staphylococcus aureus are hemolysis beta [β], while Streptococcus pneumoniae is alpha hemolytic [α].

Table 2: Results for the Laboratory Exercise of Medical Microbiology to identified the bacteria cocci gram-positive





RECOMMENDATIONS

The Microbiology requires using different experimental tests to identify the bacteria that can cause disease or infection. The procedure must be aseptic, using appropriate methods that promote the protection of the skin to prevent infection or transmission of diseases caused by these opportunistic bacteria. In the laboratory, the tests should be performed more than once to obtain reliable results.


CONCLUSION

Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae, belong to the group of gram-positive cocci bacteria, which have high importance for clinical and microbiological industry, as these bacteria are considered pathogenic, causing diseases and nosocomial infections in healthy individuals or the community. Staphylococcus aureus is a bacterium can be founded in the flora of the skin and mucosal surface. This bacterium that exhibits resistance to penicillin and can cause impetigo or other infections and disease such as bacteremia, that cause diffusion of bacteria in the blood, and endocarditis, a disease that can damage the endothelial lining of the heart. Streptococcus pyogenes is important in research, because it is easily transmitted from person to person through respiratory droplets or skin wounds by contact with an infected person, causing diseases as pharyngitis and rheumatic fever. Streptococcus pneumoniae is a bacterium which causes pneumonia, a disease in which a patient can develop lung disease. In this laboratory study the catalase test, hemolysis, Gram staining and susceptibility testing to optochin and bacitracin was performed. Through these laboratory tests, was obtained that the Staphylococcus aureus is catalase-positive, Streptococcus pneumoniae is susceptible to optochin, and Streptococcus pyogenes is susceptible to bacitracin. Furthermore, when these bacteria were cultured on blood agar, we determined that the Streptococcus pneumoniae is alpha-hemolytic, during the Streptococcus pyogenes and Staphylococcus aureus have complete hemolysis (Beta Hemolysis). Therefore, by the analysis of results, was determined that it is necessary to perform different tests for accurate the identification of bacteria causing disease or infection, to be able to assign an effective treatment. To distinguish in different genera (Staphylococcus and Streptococcus), or to identify different species in the bacterial genus (Streptococcus pyogenes and Streptococcus pneumoniae), is necessary to use different experimental methods. We conclude that a bacteria may not react equal to other in the same test, causing necessary perform several test to confirm the presence of a bacterium with accuracy.


REFERENCES

 
  1. Murray, P. & Rosenthal, K. & Pfaller, M. (2009). Microbiología Medica.209-223p.Staphylococcus y cocos grampositivos relacionados. ISBN 978-84-8086-465-7.
  2. Murray, P. & Rosenthal, K. & Pfaller, M. (2009). Microbiología Medica.224-242p.Streptococcus. ISBN 978-84-8086-465-7.
  3. Facultad de Ciencias de la Salud, Universidad del Cauca. Cocos Gram Positivos.<www.facultadsalud.unicauca.edu.co/Documentos2010/DptoMedInt/Cocos_gram_positivos.pdf> Available: November 10, 2013
  4. Gorwitz R, Jernigan D, Powers J, Jernigan J and Participants in the Centers for Disease Control and Prevention-Convened Experts’ Meeting on Management of MRSA in the Community. (2006). Strategies for Clinical Management of MRSA in the Community: Summary of an Experts’ Meeting Convened by the Centers for Disease Control and Prevention.<http://www.colorado.gov/cs/Satellite?blobcol=urldata&blobheadername1=Content-Disposition&blobheadername2=Content-Type&blobheadervalue1=inline%3B+filename%3D%22Clinical+Management+of+MRSA+in+the+Community+(CDC+website).pdf%22&blobheadervalue2=application%2Fpdf&blobkey=id&blobtable=MungoBlobs&blobwhere=1251811742663 > Available: November 10, 2013
  5. Calik S, Turhan T, Yurtseven T, Sipahi OR, Buke C. Vancomycin versus linezolid in the treatment of methicillin-resistant Staphylococcus aureus meningitis in an experimental rabbit model. Med Sci Monit. 2012; 18(11): SC5–SC8.
  6. Amador, C. Laboratorio No. 5: tinción Gram, del Dr. William Arias.<http://www.studyblue.com/#file/view/4596192  > Available: November 10, 2013
  7. NIH (National Institutes of Health). Neumonía. <http://www.nlm.nih.gov/medlineplus/spanish/pneumonia.html > Available: November 10, 2013