3 Streptococcus

3.1 Introduction
Streptococci are Gram-positive cocci that form pairs or chains during cell division. They are widespread
in nature. Some are part of the normal human flora; others are associated with major human disorders
due, in part, to streptococcal infection and, partly, to the hostś immune response. A perfect classification
system for of all streptococci has not been developed. The medically important Streptococci are S.
pyogenes (group A), S. agalactiae (group B), S. viridans (belonging to the normal flora), S. pneumoniae
(pneumococcus), etc. Enterococci were once classified as group D streptococci, but at present they belong
to a separate genus, genus Enterococcus. Streptococci are non-motile, non-sporulated and may or may
not have a capsule. In this chapter we will discuss the laboratory diagnosis of infections caused by
Streptococcus pyogenes.
Most streptococci that possess the group A antigen are pyogenic streptococci. Pyogenic streptococci
are β-hemolytic (typically producing large areas of clear hemolysis around small colonies). Pyogenic
streptococci are usually sensitive to bacitracin. Streptococci are potentially involved in a wide variety
of diseases. Pyogenic streptococci may become pathogenic due to multiplication and invasiveness. The
biological properties of the infecting microorganisms, the nature of the host response and the entry site
of the infection have a great influence on the clinical picture. Streptococcal infections could be grouped
as follows:

• Invasive diseases, in which we can include pharyngitis, streptococcal angina, erysipelas, various
upper respiratory tract infections, pneumonia, streptococcal impetigo, cellulite, necrotizing fasciitis,
puerperal fever, infectious endocarditis, streptococcal sepsis, etc.

• Diseases caused by lysogenized streptococci, in which we can include scarlet fever and toxic strep-
tococcal shock syndrome. These are toxin-mediated diseases.

• Post-streptococcal diseases including acute rheumatoid arthritis (ARA) and acute post-streptococcal
glomerulonephritis (AGN). Different authors also consider other clinical entities.

3.2 Laboratory diagnosis of infections caused by microorganisms of the

genus Streptococcus
We will discuss the laboratory diagnosis of streptococcal pharyngitis. In order to help confirm a post-
streptococcal disease, we will discuss the ASLO (Anti-StreptoLysin O) reaction.

3.2.1 The direct bacteriological diagnosis in streptococcal pharyngitis

1. The clinical specimen is represented by purulent secretion from the pharynx. The collection and
transport must be carried out in accordance with a series of rules (as close as possible to the onset
of the disease, before the patient has received antibiotics, as quickly and correctly from the point
of view of the techniques used, complying with all the rules of asepsis and antisepsis, the sampling
is preferably done in the morning before the patient eats and before brushing their teeth, without
using different mouthwash solutions, and if these conditions were not met, the sampling will be
made after at least 4 hours, etc). The sample should be processed as soon as possible, but in no
case should it take more than 2-3 hours from sampling to cultivation (preferably 1-2 hours). If we
estimate that this time limit will be exceeded, a transport medium should be used, eg the Stuart
media. In this case, the clinical specimen should be processed in no more than 24 hours.

6
 3.2 Laboratory diagnosis of infections caused by microorganisms of the genus Streptococcus

2. The microscopic examination includes the realization of two smears of the clinical specimen col-
lected and transported properly, which will be stained with methylene blue (MB) and Gram’s stain,
respectively. The smears are examined with the aid of an immersion optical microscope and the
presence of cells from the pharyngeal level, the presence of inflammatory cells (eg leukocytes, py-
ocytes) and the presence of Gram-positive cocci distributed separately, in pairs or in chains, as well
as other types of microorganisms are noted. The microscopic examination of the c.s. has only an
orientative role.

3. Cultivation on culture media of the clinical specimen is performed in such a way that isolated colonies
and a pure culture can be obtained. These will be the basis for identification. Pyogenic streptococci
are pretentious germs that do not develop on ordinary culture media. The most commonly used
medium is blood agar, on which the culture appears in 18-48 hours, at 35-37 ◦ C. If we do not notice
the appearance of characteristic colonies after 24 hours, we re-incubate the Petri dish for another
day. The colonies have an S-type appearance with a diameter of 0.5-1 mm, surrounded by an area
of β-hemolysis (an area of clear hemolysis, with a diameter much larger than the colony diameter).
During streaking, at least in one of the sides of the "polygon", we must make "a stab" in the media
so that the inoculum is seeded within the media. While there are other technical variants, this
maneuver is necessary to detect the activity of hemolysin O (this streptolysin is inactivated in the
presence of oxygen). Species that produce capsular material, made up of hyaluronic acid, often give
rise to mucoid (M-type) colonies. Selective media may also be used for cultivation (eg blood agar
with addition of trimethoprim-sulfamethoxazole).

4. The identification of the pathogenic microorganism involved will be based on several characteristics:
• Morphological and staining characteristics: They are Gram-positive, spherical or ovoid cocci,
with a diameter of about 1 µm. They divide in such a way that they form a perpendicular
plane to their long axis and are thus usually arranged in chains. The length of the chains vary
and is conditioned by environmental factors.
• Culture characteristics: They produce S-type colonies with 0.5-1 mm diameter, surrounded by
an area of β-hemolysis, although M-type colonies may also be present.
• Biochemical characteristics:
- Pyogenic streptococci produce β-hemolysis.
- The PYR test identifies the synthesis of pyrrolidonyl amidase (currently there are rapid
commercial tests for the PYR assay).
- Multiplication of group A streptococci is inhibited by bacitracin (antibiotic produced by
Bacillus licheniformis). It is estimated that less than 1% of Streptococcus pyogenes strains
are resistant to bacitracin.
- Pyogenic streptococci are resistant to trimethoprim-sulfamethoxazole.
• Antigenic characteristics:
- Commercial tests can be used for the rapid detection of the specific polysaccharide group A
of pyogenic streptococci in c.s., after chemical or enzymatic extraction (eg with pronase).
The techniques used may be indirect agglutination (latex agglutination), coagglutination
or ELISA.
- Through slide agglutination reactions (latex-agglutination, coagglutination), or through
precipitation reactions, the (Lancefield) group or streptococcal antigen type can be deter-
mined.
- S. pyogenes (Lancefield group A) is divided into serotypes based on M protein antigens
(over 80) through capillary tube precipitation reaction and T (28 serotypes) by the slide
agglutination reaction. These tests are performed in reference centers.
- Other tests used in identification: Nucleotide probes may be used for the direct detection
of group A streptococci in the pharyngeal exudate.

7
3 Streptococcus

5. Pyogenic streptococci maintain their known sensitivity to penicillin (although ¨tolerants̈trains,

which are inhibited but not destroyed by penicillin, have been recently identified). For patients
allergic to beta-lactams, erythromycin may be selected for treatment, but strains resistant to this
antimicrobial have been identified and, in this case, the antibiogram becomes necessary. In general,
the antibiogram is performed for epidemiological purposes.

3.2.2 Serological laboratory diagnosis

The immunological diagnosis of the infections produced by the group A beta-hemolytic streptococci
could consist of the investigation of either the humoral immune response (the presence and the antibody
titer, within the serological diagnosis) or of the cellular immune response. Next we will discuss only the
serological diagnosis, which is of practical importance.
Serological diagnosis is useful for certifying the etiology of post-streptococcal disease (ARA, AGN),
identifying a possible hypersensitivity state, retrospective diagnosis of a streptococcal infection, evaluating
the clinical evolution and the effectiveness of the treatment. Serological diagnosis is usually made through
the Anti-StreptoLysin O (ASLO) reaction, which identifies titers of anti-streptolysin O antibodies. Testing
is done dynamically, on sera collected at intervals of 7 - 10 days. For our geographical area, a title of
200 (maximum 250) ASLO units is accepted as normal. Internal and external quality control is strictly
necessary.
There are serological tests to determine the presence and titer of antibodies raised against antigenic
structures (streptodornase, hyaluronidase, streptokinase). The titer of anti-streptodornase antibodies is
increased in patients with skin infections and should be investigated if acute glomerulonephritis is sus-
pected. Anti-carbohydrate (passive hemagglutination) or anti-MAP (via latex agglutination) antibodies
may also be determined.

8
4 Pneumococcus

4.1 Introduction
Streptococcus pneumoniae are Gram-positive, elongated, lancet-shaped (teardrop-shaped) cocci, gen-
erally arranged in diplo (two by two) on the longitudinal axis; they are encapsulated, non-sporulated,
immotile. Pneumococci are aerobic, facultatively anaerobic. They produce α-hemolysis (incomplete
hemolysis) on blood agar, similar to Streptococcus viridans. Their growth is favored in an atmosphere of
5% CO2 at a temperature of 37 ◦ C. Streptococcus pneumoniae can become pathogenic through multipli-
cation and invasiveness, leading to various infections of the upper and lower respiratory tract, the middle
ear, sinuses, but other infections caused by hematogenous dissemination (eg meningitis, endocarditis,
etc). Pneumococcal pneumonia is accompanied by the presence of alveolar edema and fibrinous exudate,
followed by the appearance of red blood cells and leukocytes.
Due to the fact that it can be part of the normal microbial flora, there are a number of problems
regarding the interpretation of the significance of the presence of pneumococci in clinical specimens that
can be contaminated with this flora (eg sputum collected through cough and expectoration). One of the
major therapeutic problems arising is the emergence of penicillin-resistant pneumococci as well as the
development of antibiotic resistance to other antibacterial drugs.

4.2 Laboratory diagnosis of infections caused by Streptococcus

pyogenes
In order to discuss the laboratory diagnosis of pneumococcal infections, we will choose pneumococcal
pneumonia.
The diagnosis of pneumococcal pneumonia is clinical, radiological and bacteriological (direct) in order
to establishing the etiology.

1. The collection and transport of the clinical sample must be carried out in accordance with a series
of general rules (as close as possible to the onset of the disease, before the patient has received
antibiotics, as quickly and correctly from the point of view of the techniques used, complying with
all the rules of asepsis and antisepsis etc). Depending on the disease caused, the following clinical
specimens can be collected: sputum, bronchial or tracheal aspirate, pharyngeal exudate, otic or
conjunctival secretions, pleural fluid, pericardial fluid, CSF, pus, blood, necroptic material. In
pneumococcal pneumonia the etiologic agent can also be isolated by hemoculture. Next we will
discuss the case where the c.s. is represented by sputum.

2. The microscopic examination of the clinical specimen includes the preparation of at least two smears
of the clinical specimen, collected and transported properly, which will be stained with methylene
blue (MB) and Gram’s stain, respectively. The smears are examined with the aid of the optical
immersion microscope and we note the presence of:
- cells from the alveolar level (eg alveolar macrophages, also known as dust cells),

inflammatory cells (eg leukocytes) and

- Gram-positive, elongated, lancet-shaped cocci, generally found in diplo, encapsulated (with a
common capsule), unsporulated.
Specifically for the examination of sputum, the microscopic examination is used to certify the quality
of the clinical specimen (eg. the Murray-Washington scoring system) and make a presumptive
identification of the microorganism involved. In methylene blue staining, the capsule may appear

9
4 Pneumococcus

as a halo around the pneumococci. Using anti-capsular antibodies, a rapid identification may be
achieved, including directly on the clinical specimen (eg sputum), through the capsule swelling
reaction.

3. Cultivation on culture media of the clinical sample is performed in such a way as to obtain a pure
culture with isolated colonies, which will be identified. Pneumococci are fastidious germs, which
do not develop on simple media. They are aerobic, facultatively anaerobic. On blood agar, they
form S-type or M-type colonies, surrounded by an area of α-haemolysis (like Streptococcus viridans).
Multiplication is favored in an atmosphere of 5% CO2 , at a temperature of 35-37 ◦ C. Liquid culture
media are turned homogeneously cloudy. The clinical specimen can also be inoculated in sensitive
laboratory animals (in white mice, for example). These will act as a "selective culture media",
permitting the growth and invasivity of pathogenic S. pneumoniae. The animals are thereafter
sacrificed and a pure culture of pneumococci may be obtained through blood culture.

4. The identification of the microorganism will be based on several characteristics:

• Morphological and staining characteristics: They are Gram-positive, elongated, lancet-shaped,
generally in diplo, encapsulated (with a common capsule around two microorganisms), un-
sporulated.
• Culture characteristics: S-type or M-type colonies are formed on blood agar, surrounded by an
area of α-haemolysis (like Streptococcus viridans).
• Biochemical characteristics:
- Glucose is the main energy source for pneumococci. They develop glycolytic, proteolytic
and lipolytic enzymes.
- Inulin (a carbohydrate) fermentation is an important biochemical characteristic, useful in
differentiating pneumococci from S. viridans (there are S. viridans strains that ferment
inulin). This is done with the use of a medium which contains inulin and a pH indicator.
In the case of a positive reaction a change of pH will lead to a color change in the media.
- Pneumococci develop autolytic enzymes. Autolysis is induced and accelerated by bile,
bile salts, bile acids; the test is useful in identifying pneumococci (Bile lysis test, Neufeld
reaction).
- Pneumococci are also susceptible to optochin (ethyl hydrazreine), the susceptibility to
this substance begin also useful in the identification and differentiation from Streptococcus
viridans.
• Antigenic characteristics
- The most important antigenic and pathogenic determinant is the capsular polysaccharide
(the K antigen), which protects the pneumococcus from phagocytosis and allows invasive-
ness. The structure of the capsular polysaccharide is specific to each serotype. To date,
over 85 different capsular serotypes have been identified, the structure of which has been
determined for most serotypes. Specific polyvalent and monovalent anticapsular antibodies
have been produced, useful in the identification of pneumococci by means of the capsule
swelling reaction. The agglutination reaction can be used for the same purpose.
- Due to the fact that large amounts of Ag K are eliminated through the urine, its pres-
ence can be evidenced by latex-agglutination reactions, through immunoelectrophoresis or
through commercial lateral flow immunoassays.
• Other useful tests for identification: Nucleotide probes can be used to identify the rRNA.

5. The antibiogram is mandatory and is usually performed by diffusimetric methods on medium sup-
plemented with defibrinated sheep blood. Disks with oxacillin (1 mg) are used to check the sus-
ceptibility / resistance to penicillin. In case of serious infections, the diffusion antibiogram must be
complemented by other determinations.

10