Pneumococcus

The terms pneumococcus and Diplococcus pneumoniae have been now replaced in
Bergey's Manual by Streptococcus pneumoniae although all three designations
are in common use. First isolated in l88l by Louis Pasteur in France the
Streptococcus pneumoniae has provided us with a model for genetic
transformation between cells and for the role of antigenic determinants in
distinguishing various types within a genus. The reason for such intensive
study of this pathogen is attributed to its ability to cause pneumonia in
humans.

Morphology

The pneumococcus is typically a small, slightly elongated coccus, one end of
which is pointed or lancet shaped. Occurring in pairs commonly, chain
formation will develop on artificial media, especially those with a low
magnesium in concentration. Sometimes, oval an elongated bacillary forms are
found. The pneumococci are non-mobile and do not form spores. However, they
do have well- defined capsules in most strains which are readily demonstrated
in milk, blood or serum cultures. Aniline dyes stain the pneumococci which
are also gram positive in young cultures (24 hours) and gram negative in
older cultures. In stained preparations the capsule may appear as an
unstained halo around the cell as with India ink. The best method for
showing capsule is the Neufeld-Quellung Reaction which binds homologous
antibody with capsule polysaccaride rendering it refractile.

Physiology

Some types of pneumococci grow on nutrient media while others do not. There
are semi-synthetic media that have been developed based on gelatin or casein
hydrolysate and supplemented with vitamins and amino acids. Choline,
nicotinic acid and panthenic acid are general requirements as well as some
types needing biotin and ascorbic acid (vitamin C). Non essential compounds
such as purines and pyrimidines may be supplied to enrich the medium.
Sedimentation of encapsulated pneumocci occurs in liquid media, such as
litmus milk, only after it has been acidified. Pneumococcal cultures grow
between 25o and 42o with a pH of 6.5 to 8.3.The optimum pH is 7.8. Although
all of these requirements are necessary to culture this bacterium they do not
always resemble in vivo conditions. For example, in sputum, pus, serous
fluid and body tissues they are found in small chains not pairs.

Pneumococci are facultative anaerobes and, in general, can ferment sugars,
producing large amounts of lactic acid and smaller amounts of valatile acid
and ethanol. They also ferment insulin which is a diagnostic feature of
pneumococci. Care must be taken when studying museum strains since they have
lost the ability to ferment insulin.Capsule growth can be stimulated by
glucose providing sodium hydroxide is used to neutralize the lactic acid
produced.

S. pneumoniae & Alpha Hemolytic Streptococci

Streptococcus pneumoniae commonly called pneumococcus or diplococcus, is the
commonest cause of lobar pneumonia in adults.

On blood agar the pneumococcal colonies are transparent mucoid colonies that
later become shaped like a checker. Alpha hemolytic streptococci produce a
small raised opaque colony. The pneumococci in a turbid suspension of T-Soy
broth will dissolve when a few drops of a solution bile salts or sodium
dodecyl sulfate is added. while most strains of alpha hemolytic streptococci
are bile insoluble. Most strains of pneumococci will ferment the carbohydrate
inulin but most streptococci do not. However, because S. sanguis and S.
salivarius will ferment inulin occasionally the test should be performed in
conjunction with some other procedures.

The optochin growth inhibition test is the most widely used test for
differentiation of pneumococci and alpha hemolytic streptococci. A paper
disc containing 5 ug of ethylhydrocapreine HCl (Optochin) is placed on a
blood agar plate heavily inoculated with the pneumococci. After overnight
incubation at 37oC pneumococci will exhibit a zone of inhibition greater than
l5 mm in diameter.

Taxo P discs is the trade name of BBL for optochin discs. One of the most
frequently used tests for pneumococci is based on their solubility in bile
whereas Streptococcus salivarius and Streptococcus mitis do not have this
property. Sodium taurocholate, sodium lauryl sulfate and sodium deoxycholate
are the salts used in the test. It had long been known that colonies of
pneumococci would lyse after 24 hours turning the round colonies into
checkered like structures. In l923 Avery and Cullen showed that washed
extracts of pneumococci contained an enzyme capable of lysing bacteria. The
enzyme can be inactivated at 60oC after 30 minutes, but cannot kill beta
hemolytic Streptococci even under optimum conditions. However, this enzyme
does accelerate natural autolytic processes in pneumococci and furthermore,
is most active when in the presence of a bile constituent such as sodium
deoxycholate. Although the optimum pH for this enzyme is between 6 and 8,
bile acids go out of solution at pH 6.5. Therefore, the reaction must be
carried out in a more alkaline solution, usually pH 7.6. Chlorides of
monovalent cations inhibit lysis in low concentrations such as 0.004 - l% but
may, on the other hand, accelerate lysis in high concentrations. It is now
known that this enzyme hydrolyses proteins and thus may be considered a
protease. Similarly, if pneumococci are grown with steroids such as
cholesterol, or in blood or serum, which are sources of cholesterol, the
bacteria will be saponin soluble.

Since this discussion has been on properties of pneumococci, it would be
fitting to continue now with more diagnostic tests commonly employed in the
laboratory. All types of Streptococci are sensitive to ethyl hydrocupreine,
commonly known as optochin.

However, optochin, in concentrations of l in 5,000 or stronger are required
to demonstrate sensitivity whereas penumococci will not grow within 5 mm of
an optochin disc even at concentrations of l in 500,000 to l in l00,000.
Therefore, this provides a method to distinguish between Streptococcus
pneumoniae and the other Streptococci.

Autolysis of pneumococci colonies after 24 hours is a consequence of the
absence of the enzyme catalase. The end product of respiration in this
enzyme is hydrogen peroxide which in many bacteria is broken down by
catalase. Accumulations of hydrogen peroxide are lethal causing
autosterilization of the colony. This must be a prime consideration in
storage of colonies. Pneumococci can be kept for several months in a
refrigerator at 4`C or if they are plated on blood agar or both. Remember
that erythrocytes are a source of catalase. Another way to keep pneumococci
is to inoculate them into a medium containing a reducing agent such as
cysteine or sodium thioglycollate which can remove free oxygen. Since this
bacterium is a facultative anaerobe it can continue to survive without
producing hydrogen peroxide.

Soaps such as ricihnoleate and oleate are pneumocidal in dilations of .04 and
.004 respectively. Phenol and mercuric chloride can be used for the same
purposes.

Finally, a specific feature to type 3 strains is that they stain meta
chromatically with methylene blue.

Mouse Virulence test

The white laboratory mouse is extremely susceptible to pneumococci and can be
used to isolate them from mixed culture. Fill a l ml syringe with saliva
previously collected in a small test tube and inject a mouse
intraperitoneally. If the saliva is from a carrier of pneumococci, the mouse
will die within 24 hours. Pneumococci can be isolated from the lungs of the
dead mouse by cutting open the mouse with sterile scissors, swabbing the
viscera with a sterile swab and smearing the swab over a blood agar plate.
Alpha hemolytic colonies of pneumococcus should be bile soluble, ferment
inulin and be sensitive to optochin. Another procedure used is the mouse
virulence test. One millelitre of saliva, from a patient suspected of
carrying pneumococci, is injected intraperitoneally into a mouse. If after
24 hours the mouse is alive then pneumococci were not present. But, if the
mouse dies, the abdomen is cut open and the viscera are swabbed and a culture
is made on blood agar. After eighteen to twenty-four hours tests such as
insulin fermentation, bile solubility, and optochin sensitivity are performed
to confirm the bacteria identity.

Cell Wall

Pneumoccal cell walls assume the basic structural features of all gram
positive cell walls, that is, the alternating N-acetyl-
glucosamine-N-acetylmuramic acid backbone, typical of peptidoglycan.
Tetrapeptides of two alanine, one glutamic acid and one lysine residue are
connected to the muramic acids. Cross linking occurs between the
tetrapeptides to form dimers and trimers. At irregular intervals this
polysaccharide backbone is cross linked to chains of teichoic acid polymers.
These polymers are rich in choline, galactomsamine and phosphate and probably
contain ribitol, glucose and diamionotrideoxy hexose. Gotschlich and Liu
have proposed that there are two separate polymers, teichoic acid and
peptidoglycan. The structure has not been established although fragmentation
by nitrous acid and periodate indicate a repeating structure.

Studies on the pneumococcal cell wall products after autolytic enzyme
digestion show that the major activity of this enzyme, previously mentioned,
is that of a amidase. It acts in cleaving the amide bond between alanine and
muramic acid yielding a large molecular weight choline containing fraction
and small lmolecular weight peptide fraction. This enzyme activity is the
same as that responsible for sodium deoxycholate induced lysis of whole cells
since the choline containing products of the two processes are very similar.

If pneumococci are grown in a medium containing ethanolamine instead of
choline, the ethanolamine will be incorporated into the teichoic acid
component of the cell wall. In this state the cells fail to divide normally
and form long chains or linear clones. More importantly, the bacteria become
resistant to autolysis and do not lyse even in the presence of penicillin.
Another consequence is that they lose the ability to undergo genetic
transformation (to be explained below). Therefore, there must be a close
relationship between the teichoic acid and peptidoglycan components if normal
functions are to be carried out. Specificallly, the N(CH3)3 moiety plays a
critical role in cell wall properties. Ethanolamine may cause alteration of
the enzymatic recognition site or may cause distortion of the orientation of
the wall polymers. It would seem that this would be evolutionarily
advantageous for pneumococci. Some characteristics of type specific Ags are
given in the slide. It should be noted that antigene relationships exist
between some types of pneumococci and other organisms, usually with respect
to the capsular antigen, and as stated before, between various types of
pneumococci.

Antigenic Structure

The most recent literature shows that there are approximately 82 types of
pneumococci which have been differentiated by immuno- logically distinct
polysaccharides in their capsules. These polysaccharides form polymers
resulting in hydrophilic gels on the surface of the bacteria. For example,
type 3 consists of repeating units of cellobiuronic acid, that is,
D-glucuronic acid-beat (l-4) D- glucose-beta (l-3). Type 8 cross reacts with
type 3 since it has D- glucose, D-galactose and D-glucuronic acid arranged so
as to include units of cellobiuronic acid. The biosynthesis of type 3
capsule starts with uridine triphosphate (UTP) and glucose-1-PO4 reacting to
release pyrophosphate (P-Pi).

The product is uridine diphosphoglucuronic acid (UDPGA) by UDPG
dehydrogenase. UDPGA subsequently releases glucuronicacid.

Smooth strains, those with capsules, are virulent, while rough strains,
unencapsulated, are avirulent. Virulence is also proportionate to capsular
size. Intermediate variants produce a smaller capsule and are less virulent
than type 3 fully encapsulated. Pneumococci also posses three kinds of
somatic antigens, namely C, Rand M exclusive of the polysaccharide capsule.
The C antigen or C substance, as it is usually called, is a species specific
of carbohydrate found in the cell wall. It is purified from autolysates or
dexycholate lysates of the pneumococci and contains
galactosamine-6-phosphate, phosphoryl choline, diamino trideory-hexose and
elements of cell wall mucopeptide. The C substance is not inactivated by
peptic or tryptic digestion and yields 30% reducing sugar on hydrolysis and
contains 6.l% nitrogen. Trichloroacetic acid extraction of this produce
gives the teichoic acid polymer of libitol and glucose.

The C substance can be precipitated by beta globulin in the presence of
calcium ions. The beta globulin is not an antibody but a protein in the
blood during the active phase of certain acute illnesses persisting during
the lesion phase and disappearing with convalescence.

The type-specific M antigen is important in determining antigenic
heterogeneity in non-encapsulated forms. It plays no role in virulence and
varies independently of the capsular poly saccharide. The M antigen is
soluble in acid-alcohol, is a protein and can be destroyed by proteolytic
enzymes. Along with having no antiphagolytic properties it is found in both
nonencapsulated and encapsulated forms.

Antisera against somatic antigens is useful against virulent forms although
negligible in comparison with antibodies to type specific antigens.
Recently, a species specific nucleoprotein antigen has been found deep inside
the cell. Little is known about it at this time since it is probably of no
importance. Summarizing the antigens, there is a nucleoprotein component deep
in the cell, the species specific C substance near the surface, a
type-specific M antigen near the surface, the protein R antigen and the type
specific polysaccaride or capsular antigen.

Tying of Pneumococi

There are three methods of typing pneumococci and all are variations of the
same principle. They are: l) agglutination of pneumocci with type specific
antisera; 2) precipitation of specific soluble substance (SSS), that is, the
polysaccharide haptene with type specific antiserum and 3) the Neufeld
Quellung reaction.

The Quellung (swelling) phenomenon was first described by Neufeld in l902 and
reinstated in l93l and is now in general use. The method involves mixing a
suspension of unknown type pneumococci with undiluted rabbit antiserum on a
slide. To facilitate observation, a small amount of Loeffler's alkaline
methylene blue is added to the mixture. The slide is then examined under the
microscope. In the presence of homologous antiserum the capsule swells
without any change in the bacteria itself. Heterologous serum does not
elicite the swelling. The reaction is rapid and swelling is apparent in a
few minutes. The nature of the swelling is not entirely known but may be
reversed by the addition of homologous SSS. !TOPIC TOXINS Pneumococci produce
a variety of compounds that in other bacteria are considered the primary
factors of virulence. However, they are not determinants of virulence in
pneumococci. In l934 Oram described a leucocidin, active for rabbit white
cells, in pneumococcal filtrates. Hyaluronidase is released as in the
Staphylococci. Pneumococci produce a hemolytic toxin called pneumolysin
which is related immunologically to the oxygen labile hemolysins of
Streptococcus, Clostridium tetani and Clostridium perfringens. During
autolysis, a purpura producing principle is released, causing dermal as well
as internal hemorrhages in rabbits. An interesting phenominon of pneumococci
is the relatively non toxic, type specific capsular polysaccharide into the
fluid phase. Normally, polysaccharides have no role in toxicity and even in
this cause they are, in themselves, harmless. It is their ability to bind to
and neutralize antibodies directed against the bacteria before the antibodies
reach the site of infection. Unlike Staphylococcus aureus, this bacterium
does not produce a fibrinolysin in culture and none is indicated in sections
of infected tissue. Virulence in pneumococci is due mainly to invasiveness
and is pronounced since the capsule surrounding the organism prevents
phagocytosis by macrophages. Successful destruction of a bacteerium occurs
only when large groups of macrophages can pin it against the tissue of the
lung leading to ingestion. When the macrophages are present in small numbers
they cannot phagocytize pneumococci since they keep sliding off the capsule.
If the pneumococci enter the sterile spinal fluid where there is no tissue
against which to pin them, even large numbers of macro- phages are useless
and the result of the infection is pneumococcal meningitis. In l970, Kelly
and Greiff characterized a protein released by pneumococci and found it to be
toxinogenic. It may be interesting to study their work as a model of
scientific research. In l967 they found that all clinical infections of
pneumococcal pneumonia possessed the enzyme neuraminidase, although old
cultures lost this activity. The organism was grown in basal media
containing yeast extract, tryptose, NaCl, NaHCO3 and N2HPO4. Pneumococci
were harvested and filtrates were made. They determined protein content of
the filtrate by the method of Folin-Ciocalteau and then found neuramindase
activity by measuring the quantity of N-acetylneuraminic acid liberated from
N-acetylneuranlactose. The acid was measured by the method of Warren.
Toxicity was determined by intraperitoneal inoculation in mice and the LD50
values derived by the method of Reed and Muench. The extract had an LD50 of
635 micro grams of bacterial protein and could be inactivated by incubation
at l00`C for 3 minutes. However, with pure enzyme the LD50 was 22 micrograms.
The major emphasis of pneumococcal pathogenicity has been on the
polysaccharide capsule but not avirulent encapsulated strains have been found
and conversely a search is on now to see if there exist virulent,
unencapsulated strains with neuraminidase activity. Neuraminidase splits
sialic acid residues from many substrates, especially the mucopolyusaccharide
from the glomeruli of the kidney. Future research may show this to be an
important part of pneumococcal pathogenicity.

Genetic Transformation in Pneumococci

The encapsulated forms of pneumococci are virulent while the
unencapsulated forms are avirulent. Exercises in culturing these bacteria
have shown that strains and become encapsulated spontaneously and therefore
virulent. Avery, MacLeod and McCarty studied the transformation of rough (R)
to smooth (S) strains in l944 and were able to isolate the transforming
principle. In summary Type II (R) pneumococcus was transformed to Type III
(S) pneumococcus. A virulent S strain was cultured in nutrient broth
containing human pleural or ascitic fluid. The culture was then sterilized
with alcohol which had no effect on activity of transformation. It had
previously been found that the cells possess an enzyme capable of destroying
the activity of the transforming principle.

To eliminate this enzyme the pleural fluid was heated to 60` for 30 minutes.
The resulting serum and its contents was seeded with a 5 hour blood broth
culture of R36A and incubated for 24 hours. After incubation anti R serum was
added and all the R cells precipitated to the bottom of the test tube. Thus,
any S cells would remain in the supernatant. The supernatant was plated and
the culture grown was Type III encapsulated pneumococcus. In repeating the
procedure the transforming principle was isolated from the serum and analysed
chemically and physically. The principle could withstand 65`C for 30 to 60
minutes. A positive reaction was found for the Dische test for DNA and
negative reaction for the Bial test which is sensitive to RNA. Since the
principle could be extracted with alcohol and ether at -l2` it was definitely
not a lipid. Trypsin, chymotrypsin and ribonuclease had no effect on
transformation implying that the principle was neither a protein or RNA.

Next, the enzyme that inhibits the transformation was tested. Sodium fluoride
was the only agent having an inhibiting effect and NaF is an excellent
inhibitor of DNA polymerase. Thus, it was learned that DNA can pass from S
cells to R cells resulting in encapsulated, virulent pneumococci. Actually,
the DNA codes for uridine diphosphoglucose dehydro- genase, the enzyme
responsible for producing the glucuronic molecules of cellobiuronic acid in
Type III pneumococcus.

Pneumonia

Pneumonias are diseases of the respiratory tract and will be considered in
respect to causes, effects on the body, symptoms, complications and
treatments.

Pneumonia is simply defined as inflammation of the lung, caused by bacterial
or viral infection, leading to consolidation of lung tissue. Do not confuse
this with other disorders such as mucous secretions in the bronchial tree
shown by excessive coughing and commonly known as bronchitis. Anatomically,
pneumonia can be described as lobar or bronchopeneumonia, depending on the
areas of the lung affected. Rarely, the entire lung is involved in
consolidation but it may occur anywhere in the lung. Aspiration pneumonia is
a complication of intestinal, pyloric or esophageal obstruction in which the
infection is moved from a region without disease to the lungs by vomiting.
Clinically, pneumonia appears as rigors, vomiting, plural chest pain,
feelings of cold even with a high fever, sore muscles and malaise. The
symptoms, generally, show between two days to two weeks after infection.
Some patients also suffer from abdominal pain which, on first examination,
may be mistaken for cholecystitis (inflammation of the gall bladder) or a
perforation in the intestine.

To make a proper evaluation the physician employs a variety of tools, one of
these, the chest X-ray, will show a darkened area in the upper right lobe of
lobar pneumonia and patchy shadows of irregular shape in both lungs of
bronchopneumonia. Bronchopneumonia will have more numerous shadows in the
lower lobes. There is little or no collapse of the lobe in which
consolidation lies and the hilar glands are not enlarged. If the lobe is
partially collapsed the pneumonia is secondary to bronchial occlusion, in
which case the hilar glands are malignant. But since there is consolidation
in both primary and secondary pneumonia, the origin of the lung disorder is
not discernible by X-ray.

In the case of bacterial infections, of which Streptococcus pneumoniae is the
most important, a complete blood count will reveal an increase in the
polymorphonuclear population. Frequently a sputum culture is obtained and is
sometimes a good diagnostic aid, but technical problems reduce its
usefulness. For example, saliva can get mixed with the sputum. When a wire
loop is used in transferring the sample the saliva adheres to the loop and
only bacteria in the saliva are cultured. Since some bacteria are pathogenic
in only selected parts of the body the culture may contain pathogens that
were not present in the lungs.

Even if only sputum is transferred the causative organisms may be absent due
to their irregular distribution in sputum. Lastly, if the pathogen is
present in the sputum it may still be masked by overgrowth of the culture by
a non-pathogenic bacterium. Although gathering sputum over a twenty- four
hour period would be advantageous, it is usually only taken an hour after the
patient awakens in the morning. As you can see, it is very easy to get false
readings from sputum cultures unless careful technique and awareness of the
pitfalls are used.

To prevent post operative pneumonia, a common occurrence in hospitals, a few
procedures should be followed. Preoperative medication and anaesthesia
should be minimal and always the anaesthetic apparatus should be sterile.
After surgery patients should be instructed to begin deep breathing and
coughing to help eject or dislodge any foreign material in the lungs.

Complications

Superinfection, that is, colonization by Staphylocci or Enteric bacteria, is
the most frequent complication of pneumonia. This is readily treated with
antibiotics. Tachycardia (rapid heart rate) and fever pyrexia are always
present but prolonged fever can be due to pus in the lungs (emphysema), lung
abscess, collapse or lung obstruction (atelectasis). Other problems,
especially in advanced or untreated pneumonia, are plural effusion,
pericarditis, endocarditis, pneumococcal arthritis and meningitis. Some
patients even absorb bacteria or their products into blood and tissue
(septicemia) and immediately have a fall in blood pressure. In untreated
cases, renal or hepatic failure may develop and is in fact, the major cause
of death associated with pneumonia.

Treatment for pneumonia, if the exact cause is now known, is with methicillin
or doxiacillin added to benzyl penicillin and administered intravenously or
intramuscularly. Finally, about ten percent of pneumonia patients are unlucky
enough to develop herpes simplex and cyanosis.

Pathology

In lobar pneumonia there are three stages through which the lung changes.
Initially, air spaces in the lobe fill with fluid containing some
polymorphonuclear cells and causative bacteria. Alveoli fill with red blood
cells, white cells and fibrin. Since the alveolar vessels are filled with
blood the lung looks like a liver with a red hue and is firm and airless.
This stage is called Red Hepatization.

Next, the bronchial arteries near the lobe become clogged and the lung
becomes yellowish grey. Hence, this stage is Grey Hepatization.

Finally, the bacteria disappear and over two or three weeks monocytes
phagocytize the remaining dead tissue. This is the Resolution stage.

Bronchopneumonia has the same stages but in smaller more abundant areas. It
may be noted that tissue necrosis is greatest when Staphylococci are
secondary invaders to pneumococci. Up to this point the discussion has been
anatomical. Recent advances in microbiology and immunology have made
classification of pneumonia by the causative organism possible. Although
there seem to be as many causes as there are bacteria, the major cause is
Streptococcus pneumoniae. Post operative hospitable pneumonia has seen a rise
in Staphylococcal and Klebsiella type pneumonia.

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