MYCOPLASMA

Tne discoveryof Mycoplasma goes back to the 19th century (1898), when two
anch scientists studying the pleural fluids of cattles suffering from a disease
led bovine pleuropneumonia, discovered some microorganisms that were
waly from the ones known before. The disease was highly contagious
different

nd became widespread in Europe during the 19th century. The disease causing
organisms were different from bacteria
and were given the name
pleuropneumornia organisms
like
(PPLO).These PPLO were aerobic and
could be cultured on a rich organic
medium and were given the name,
Mycoplasma mycoides. The first
(0)
(c) cultured forms were some times
spheroidal in shape, but also produced
a wide variety of minute granular
structures, thin branching filaments,
asteroid structures or many irregular
forms. Due to the mycoplasma
this,
were called pleomorphic.

(b) (d) After the discovery of PPLO from

initial

bovine serum, similar pathogenic forms

were reported from a wide variety of
Fig. 9.1Mycoplasma veterinary sources like sheep, goats,
Colony
morphology and cell shapes of a

mycoplasma. (a) entire colonies (b)

dogs, rats and In animals, they
mice.
longitudinal sectionof two colonies witha variety of
(PPLO)are associated
9rowing on
agar surface (c) spherical form
() irregular filamentous
pathological conditions such as,
form.
rheumatic or arthritic diseases,
 Text Book of

258 Microbiology

tract and adjacent tissues

infections of mammary glands,respiratory and

of urinogenitial system.
inflammation
known to be pathogens. The
human
PPLO are also
Some the hurman
of
flora consists of [Link] causing primary
mycoplasms (PPLO) atypical
to any bacteria that generally
is not attributed
(atypical pneumonia Cause

pneumonia))Pneumonia.
1967) on mycoplasma indicatedthat
Subsequent researches (Doi et. al,
they
to plants. Plant diseases of mycoplasma origin
also cause disease are-asster

witches broom, dwarf disease mulberry, spike

of
yelllow, potato disease of

sandal etc.,

Many mycoplasmas are also found in saprophytic conditions in decaving nreset

matter, sewage etc.

GENERAL PROPERTIES OF MYCOPLASMA(PPLO)

The following are the general properties of mycoplasma.

1. They are the smallest, free living organisms (about 125-250 nm in

diameter).
2. They can easily pass thorough bacterial filters.

3. They are highlypleomorphic - branched, beaded, swollen, ringed or even

nen stellate. They may even be spherical, ovoid, or filamentous (branched).
hn Sometimes they resemble the hyphae of fungi in their morphology.
4. They a lack cell wall. The outer most boundary is a three layered plasma
membrane.
5. They are either parasitic or saprophytic.
6. They can be cultured in a cell free medium with an abundance of organlk
matter.
7. They are aerobic as well as
anaerobic.
8. Some members (Mycoplasma) others
require sterol for growth, while

(Acholeplasma) do not require it.

9. Some mycoplasma called 'T'strains require

urea and
a lower pH for growth than [Link] require
10. Ribosomes are present of true
in the cell, but different from those
bacteria.

11. Mycoplasmas are resistant to

penicillin but sensitive to tetracyclins.
take
12. Specific details may
of reproduction are not known. Reproduction,
place by elementary bodies
budding or binary fission.
13. Mycoplasmas are gram negative.
14. Spores, flagella etc. are absent.
 259

Mycoplasma

Classificationname for mycoplasma was PPLO, as they resembled the

original
The agent in some respects. Their varied morpholigical nature,

shape, or occasional branched filamentous structure and mold like

pleuropneumonia

gpheroidal
indicatedthat they have affinities with the group Actinomycetales,
appearance mold like bacteria. However, their taxonomic position was much
includes
that
debate. They were also classified under class Schizomycetes(Bacteria),
under
order Mycoplasmatales, family Mycoplasmataceae and genus Mycoplasma.

doubts
persisted about the taxomic affinities of
mycoplasma with bacteria,
Since

Committee on the bacterial nomenclature constituted

[Link] International
and offer
ubcommittee to goaspects of mycoplasma systematics
into all
deliberated and gave
aitable recommendations. Accordingly, the subcommittee
the following suggestions.

as stabilized L-L forms of bacteria

i) The mycoplasma are not to be regarded
(L forms of bacteria do not have cell walls like mycoplasma).

Considering their
as different from bacteria and viruses,
unique characters
(i)

the rank of a class - Mollicutes along

the mycoplasms should be accorded
with bacteria (Schizomycetes).
two orders and
Mycoplasmatales
The class Mollicutes is divided into
and
each with a single family- Mycoplasmataceae
Acholeplasmatales
belonging to each family.
ACholeplasmataceae. There are only two genera, one
(Edward and Freundt, 1970). In addition
iese are mycoplasma and Acholeplasma
are also included.
twO genera, two more
genera of uncertain affinities
wUese

hese are - Spiroplasma and Thermoplasma.

The main criterion for the distinction

between Mycoplasmataceae and

is the
Acholeplasmataceae
for growth in
requirement of sterol

the former, and its non requirement

in the latter.

are also classified

The mycoplasma
on their
into three groups based
properties. These are :
physiological

- non
(i) Carbohydrate fermenting
sterol requiring

(ii) Carbohydrate fermenting

sterol requiring
fermenting -
Fig. 9.2 (ii) Non carbohydrate
egg Mycoplasma
appearence of Mycoplasma non sterol requiring.
colony
under
microscope
 Text
Book of
260
Microbiology

above, acid is produced

from sugars
In the groups i and ii such
etc. The non fermenting species utilize as
fructose, mannose glucose,
amino
acids as carbon and energy sources (eg. Mycoplasma acids and
fatty
arthritidis).
General characters of families
Mycoplasmataceae
1. They can grow only when steroladded to the
is
medium.
2. They can grow in a temperature range of 22°C to
41°C with
being 36°C to 37°C. the
optimum
3. A pH of 7.0 is ideal for their growth.
4. The GC content of DNÀ is 23-40%.
5. They are mostly animal pathogens
(includinghuman
pathogens. beings), rarely
plant

Acholeplasmataceae
1. Members saprpophytic, presentin
sewage; sterol not
2. Require required for
a
temperature range between 37°C to growth.
65°C with
59°C.
optimum being
3. Acidic pH range is ideal (1-2).
4. They form typical "fried
egg" colonies on the
5. The GC content of DNA is 25-26%. medium.

Cell Structure
(Morphology and fine structure)
Mycoplasma cell cultures show short or long branching
spherical coccal bodies. filaments with many
The morphology of the cell depends on the nutritive
properties of the growth
medium particularlythe ratio of
to unsaturated ones. This saturated fatty acids
factor particularly affects the
membrane structure
of the organisms.

The size of the cell is about 125-250nm. It is from cells of this size which is

minimal to the existence of the organism further

growth begins. Some
mycoplasma may reach a size of 0.5 to 1.0 um.

The cell has no cell wall and there are no locomotor [Link] lack ot Cell

wall is responsible for its fragility and plasticity with which it can assume

various shapes. The outermost boundary of the cell is a plasma membrane

which is about 8-15 nm in thickness. The membrane is triple layered with tO
like
electron dense layers separated by a translucent layer. In some species,

Mycoplasma pulmonis, M. etc surface spikes are present

gallispacum n
up
is made
membrane resembling Myxoviruses. The electron translucent layer of

made up
of long chain of fatty acids while the electron dense part is
in
present
proteins and carbohydrates. Unlike in the bacterial cell, the lipids are
lipids
of the
the membrane are either cholesterol or carotenol. Some
serologically
glycolipids and the sugar moities of these molecules are important
 261
Mycoplasma

determinants
of antigen reaction in fixing and neutralízing the antibody

reactions.

membrane polyopeptide chain (protein), which is either external or

The
also contributes to the antigen reaction.
interwoven

of a protective cell wall imposes special responsibility the cell

The absence
a

membrane to maintain the

Lipoprotein
Membrane integrity in the face of varying
Ribosome external osmotic pressures.
Cholesterol interspersed with
DNA
phospholipids is known to play

Metabolite
a role in the maintenance of

membrane integrity. The

Soluble protein
absence of cellwall offers
immunity to mycoplasmas
from the action of antibiotics

Fig. 9.3 Mycoplasma like pencillin and cycloserine

A profile of mycoplasma cell which act on the process of

cell wall synthesis.

The mycoplasma membrane is also the site of many metabolic reactions involving
enzymes (which are membrane bound), and transport mechanisms. In

Mycoplasma pneumoniae,there is a specialized area on the membrane called

the 'foot' which attaches tothe animal cell surface. A needle like structure is
inserted into the cell through which mycoplasma enzymes enter and digest the
host cell
metabolites.

Ihe cytoplasm of the mycoplasma does not have an internal membrane system
le,there is no endoplasmic reticulum. There is no well defined
mesosome
Mke in bacteria. In M. gallisepticum, membrane bound vesicles or blembs
de tound whose function is not yet clear. The cytoplasm
has only two organelles
DOSomes and nuclear material.

e nbosomes are of the 70s variety and as in bacteria, protein synthesis in

se is affected by antibiotics like puromycin,streptomycin, erythromycin,
chloramphenicol etc. This aspect is of a considerable significance in the
Tapeutics. It is worth noting that cyclohexamide, an inhibitor of
protein
synthesis in 80s
ribosomes, has no effect on mycoplasma.
The
nuclear
material is fibrillary in form and is either centrally placed or
dlspersed. The DNA is naked and has no protein associated with it. The total
Content of
DNA in a mycoplasma cell amounts to between 1.5-4% of the dry
Weight of
the cell. Circular double standard DNA has been extracted from six
262 Text Book of
Microbioloqy

species of
mycoplasma. Molecular wt. ranges from 444 x10° to 1200 x 10
daltons. The GC (Guanine/(
/Cytosine) ratio varies between 24-41% in
different
species. In some cases G/C ratio is used as a taxonomic criterion.

Reproduction
No sexual reproduction has been reported. It is doubtful whether mycoplosme
can undergo congugation of the bacterial kind as it involves the cell woll
Asexual reproduction is by means of simple fission or formation

of
elementary
bodies.

Very minute bodies called elementary bodies or minimum reproductive units

are commonly formed inside the adult cells of mycoplasma. These elementary

bodies range in size from 100 300um. They can pass through minute
-

bacterial filters but are viable on culture media. These minute bodies are
often mentioned as examples of minutest independently living (viruses ar

nt independently living) entities. The elementary bodies represent the

young stage in the life cycle of mycoplasma. They enlarge to form long
filaments, spheroidal cells, chains of bead like cells etc. The elementary
bodies are released from the larger cells by the rupture of the outer
membrane.

There are also reports, of binary fission and budding in mycoplasma, as in

yeats.

Culture of Mycoplasma
Mycoplasma can be cultured in the laboratoryusing various media. The choice
of a medium depends on the species to be cultured. Mycoplasma are cultured
best on solid or semi solid agar media with a rich meat base and a high

supplement of animal protein. They (mycoplasma) do not seem to prefer

liquid media, as the growth of the colonies is not profuse on liquid media.

PH of 7.8 is ideal for the growth of cells. Yeast extract (25%), is another

useful ingredient to be added to the medium especially to culture animal

pathogens. Serum (25%), is another important ingredient in the medium.

Some the T' strain mycoplasmas require urea. Either this can be directly
of
added or the T' strains obtain it through the serum. Most of the specles
aerobes or faculatativeanaerobes, but some of them require microaerophilic
conditions and C0,.

Incubation at 37°C is prefered by But some

most of the mycoplasma.
Acholeplasma grow best at 22-30°C.
The addition of
unsaturated fatty growth
acids to the medium encourages
and for all parasitic species, cholesterol or a related sterol is essential.

Mycoplasma may also be readily cultivated in living chick embryo

 263

apasna
media which are widely used for mycoplasma culture are given
of
types
TvO

belo. as
Agar: PPLO Agar or BBL mycoplasma agar is to be prepared

1. Mycoplasmaof the brand. Melt the to 45°C: to 7 parts

of
agar and cool

it
instruction 25% yeast extract.
per
serum and one part of
2 parts of untreated horse
add
this
be between 7.6-78.
PH should
The
beef infusion broth is taken and
broth : PPLO broth or heart

2. Mycoplasma and 20% horse serum. PH is adjusted

extract solution
this is added 10% yeast

to 7.6-7.8 and identificationof

lsolation

mycoplasma from cultures.

containing
The sample (source)
be filtered
mycoplasmas are
to

through membrane filter (pore

a

size 200-450nm).Most mycoplasma

pass through the
will
and viruses

filter while other micro organisms

are held back.

be taken in a
The filterate should
sterilized
platinum loop (previously
on the
by flame) and streaked
agar. Only
prepared mycoplasma
grow on the agar
mycoplasma will
though viruses
as it is cell free. Even
cannot form
may be there, they
hosts.
plaques as there are no living

Detection of mycoplasma
in
can be identified
Mycoplasmas
following three
the culture by the
formation,
methods- Colony
Staining and
antigenic analysis.

Colony formation
of mycoplasma are
Colony features
and these are used
highly specific,
them.
Fig. Mycoplasma
9.4 toidentify and characterize on the
Mycoplasmamycoides, are formed
electron micrograph, Minute colonies
gold shadowed
nature of aar medium
by mycoplasma. After
To show branching
strain colonies start
a filamentous incubation,
the initial
in
two days and
appearing within
 264 Text Book
of
icrobiology

about a week's time the colonies attain a size of [Link].

Colonies
may be
observed visually under a dissection microscope.

Colonies appear uniformly granular with or without a dense centre.

The
colonies
also have spreading borders showing thetypical "fried egg"
[Link]
nature colony shows a dark central portion surrounded by
pleomorphic or
spherical bodies. While the fried egg appearance is most characteristi
mycoplasma colonies, some may not exhibit this character.

Another characteristic feature of mycoplasma colonies is the embedded cent.

i.e, when the colonies begin to grow, the cell are drawn into the
spaces
between agar gel, and the colonies form a ball like structure and sores
outwards. As a result, colonies can not be easily separated from the medium
by usual microbiological practices.

Staining alin

Aniline dyes are most suited for staining mycoplasma cells. Dienes' stain

(discovered by dienes, one of the first investigator of L bodies. Dines' stain can
be prepared by dissolving 2.5 gm of methylene blue. 1.25gm. of azure lI, 10.0
gms of maltose and 0.25gm of sodium carbonate in 100ml of distilled water.

This is the stock solution from which dilutions are to be used for actual stainig. tera

Cut a small block agar containing mycoplasma colonies and place it on

of the

a slide. Gently apply a few drops of stain on to the colonies with a cotton

sitior
applicator. Most of the mycoplasma colonies stain with a dense blue centre and tolo

a light blue periphery. Bacterial colonies also are stained initially, but within
Untes
about 15-30 minutes they get destained (de colorized), while mycoplasmas thc
retain the colour.
Kenc

Visual observation of mycoplasmas may also be conducted with the help of

phase contrast, or dark field microscope. Ultrastructural details. however become

obvious only under electron microscope.

Antigenic analysis
Until recently, the identífication and species classification of mycoplasma Was
based mostly on colonial morphology and biochemical reactions. In recent
years, however, systematicserological studies supplemented by techniques o

molecular biology have been of additional help in mycoplasma identificatioll

In stable suspensions, agglutination has been used for antigenic classiticau

and for measuring antibodies in naturally and experimentally infected hosts.

on
Mycoplasmas differ from a great majority of bacteria growth
in that their
solid media is inhibited by potent antisera.

Immunofluroscence of colonies of results,

mycoplasmas also yield species specific
and are particularly useful in detecting species.
mixtures of mycoplasma
 265

Moplasma

techniques, however, are complicated because of the tendency of

seriological to absorb and hold on to denatured globulin and other constituents

mycoplasma rich medium in which they are grown.

the serum
fom
the more recent molecular biological techniques include comparision
of
Some of unknown with the established species. Similarly, DNA
of
G/C ratios
between the known and the unknown is another useful method
hybridization Another molecular biological technique is to Compare the
of identification.

banding on polyacrylamide gel between species. Classification and

protein
of species by this method correlates well with the results from
identification
and hybridization
and antigenic analysis.
nucleic

Mycoplasma
and L forms
such as the presence of antibiotics like
lder various cultural conditions,

cycloserine, bacitracin etc. cell wall synthesis in bacteria is affected.

onrillin,

happens some instances (with

her the bacterial colonies die, or as it is

of the medium) they survive in the form of

citable osmotic composition
with modified cell wall or no cell wall. They
nheroplasts or protoplasts
forms of
multioly parent bacterium and survive. Such wallless
as variants of
(do no revert to
hacteria are called L forms (L phase). They may be stable
forms under suitable
normal from), or unstable (revert to normal wall

Conditions ).

have a striking similarity with mycoplasma

The colonies of L forms of bacteria
wall. Originaly mycoplasmas
Colonies possibly because both of them lack cell
however, a number of
were thought to be L forms of bacteria. There are,

differences between the two.

Mycoplasma
[Link]

Minimum reproductive unit smaller

Minimal reproductive unit
1.
larger

Filamentous forms are frequently

2 Filamentous forms are rare.
seen.

No wall.
E Retain in the biochemical
machinery required for wall
synthesis at least in part.

Cholesterol required (except

Cholesterol is not a must
for Acholeplasma
growth
Pathogenic (not - all)
Normally non pathogenic

Nuclei and base ratio resembles

Does not resemble
that of
bacteria
266 Text
Book of
Microbiology

Economic importance
great concern to human
Mycoplasma are undoubtedly, of beings, asthey
are
known to cause many human, animal and plant diseases.

Human mycoplasma
The human
mycoplasma consists
of about 8 or 9 species. Some
of these are M. hominis,M.
Salivarium,
[Link],
[Link], [Link].
[Link], T. straits etc. 0f
all these M. Pneumoniae is the
most important, while M

hominis may function as a low

grade pathogen in certain
circumstances.
Fig. 9.5 Mycoplasma
Colonies in agar of Mycoplasma M. Pneumoniae causes atypical
homintis.

Photographed unstrained,with the low pneumonia. This was first

power of the light microscope. discoveredin 1961, by Maromion

and Goodburn. Clinically,

[Link] exhibits

symptomsof febrile bronchitis,

myalgia, headache, sore throat

and cough.

Human infections from [Link] have been reported from northern

temperate zones and Australia. While atypical pneumonia affects algae groups;

those between 5-15 years are specially vulnerable.

Clinically, [Link] can be identified by the culture of throat swabs.

Treatment for the disease involves treatment for 2 3 weeks wither with

tetracyclin or erythromycin.

[Link] and T strains of mycoplasma are known to be involved n

urinogenital infections, even though reports are not very confirmatory.

some cases atleast, ovarian abscess, salpingitis and haemorrhagic cystitis a

traceable to [Link].

Animal Mycoplasma
Since
Bovine pleuropneumonia was the first
mycoplasma disease discovered.
in goat,
then PPLO (mycoplasma), is known to cause a number of diseases
tracts
sheep, rat, mice etc. Respiratory tracts, mammary glands and genital

are infected.
 267

MCoplasma

the use of suitable antibiotics liketetracyclin erythromycin

involves
Teatment

plant
Mycoplasma have
of plants harbour mycoplasmas. They (mycoplasmas)
number
Alarge
in electron micrographs of plants suffering from corn stunt,
detected
been
other diseases. Leaf hoppers are known to act as vectors
and
yellow
mycoplasma from the infected to the healthy ones. Many
Bster plant

been confirmed to
were thought tobe viral inorigin have now
transmitting
re which

diseases caused by
caused by mycoplasma. Some of the important plant
are
mycoplasma

1. Aster hellow

Potro witches
broom
2.

3. Sandal spike

4. Corn stunt

5. Dwarf disease of mulberry

6. Blue berry stunt

7. Little leaf of Brinjal

8. Western x disease

mentioned above, 'Sandal Spike' is the most important,

Anong the diseases
our forest trees. The heart
is one of the most valuable
of
as the sandal tree
oil
of this semiparasitic tree yeilds the world famous essential
WOod

sandalwood oil.

sandal tree are of two

e aisease symptoms caused by mycoplasma
in the

bpes-witches' broom and pendulous spike.

TChes'broom, the leaf area is drastically reduced, branches get stunted

broom. pendulous spike, the infected shoot becomes

e appear like a In

the apical bud and suppression of

Unusually
long due to the overactivity of

y Duds. The affected shoots droop down and hence the name, pendulous
spike.

The
is transmitted from the infected to healthy plants
pathogen (mycoplasma)
through albimalenta, JasSus indicus etc.
insect vectors, such as Moonia

bisease fully. The Forest Research laboratory

control has not been achieved
and control of this disease since
Bangalore has been engaged in the study
along and
time. Treatment includes combined therapy of fungicides antibiotics

Benlate, A wound is made in the affected tree

the terramycin or Ledermycin).
base of the stem and a paste of medicine (antibiotic + fungicide)is
 268 Text Book of
Microbioloqy

applied,and the bark replaced over the wound and tied with an adhesive
tape. Severaltreatments of this kind with a gap of not more than 100 dave
are required, to bring about relief.

Mycoplasma viruses
A number of viruses are known to infect mycoplasma. These are called
mycoplasma phages. The first mycoplasma phage
was discovered in 1970 by
Gourlay, and since then more than
75 viruses of mycoplasma have been
reported. Three categories of
mycoplasma phages have been recognised.
These are - Naked bullet shaped particles,
spherical, sheathed particles and
polyhedral particles with tails. Most
of the mycoplasma phages
obtained from have been
Acholeplasma laidlawii. All these are DNA
shaped viruses. In bullet
viruses, DNA is single stranded. In the other two, DNA is
stranded. dout