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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
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