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Basic Microbiology.

The central principle of infection control is that of control of the growth of micro-
organisms which potentially cause us harm. To underpin the concepts of infection
control it is useful to understand some basic microbiology regarding the types of
organisms involved, how they may grow and multiply, and the kinds of diseases they
may be responsible for.

Basically there are three groups of micro-organisms:


• Bacteria
• Fungi
• Viruses

Bacteria.
There are many different types of bacteria in existence, and by far the greater
percentage of them are harmless to us. Bacteria are also essential to our daily lives,
ensuring that we remain healthy, helping the development of drugs, and also in the
development of new foods.

Bacteria can be grouped visually, depending upon their shape and arrangement :
• Cocci – all round or spherical
• Bacilli – rod-shaped
• Spiral-shaped.

(coccobacillus falls between coccus and bacillus).

Each group may be further sub-divided :

Cocci
Monococcus –arranged singly
Diplococcus –2 together
Staphylococcus –haphazard
Streptococcus –chains
Sarcina – clumps in cubes
Gaffkya –groups of four

Staphylococcus is found normally in the body in three forms-


s. aureus in the nasal passages
s. epidermis on the skin
s. albus in the mouth.

Neisseria are similar to diplococcus, but are kidney-shaped. These are usually found
in pus, i.e. N. gonorrhoea, N. meningitides.

Bacilli
Found as single rods or as chains. Single rods are randomly arranged, and may be
either long and thin or short and fat. E. coli is an example, normally found in the
bowel. Salmonella is another example.
Spiral.
Spirilla –move via flagella
Vibrio –
Spirochetes –

Structure of bacteria.

The cell membrane of the bacteria is differentially permeable, and is also the site
where enzymes occur, sited on the mesosomes. The cell wall determines the shape of
the bacteria, and bacteria can also be classified by how they take up the Gramm stain,
which is dependent upon the structure of the cell wall. This occurs because all
bacterial cell walls contain muerin, a peptidoglycan. Muerin consists of parallel
strands of polysaccharide, cross-linked by short polypeptide chains to give a rigid
structure, forming a net around the bacteria.

In Gramm positive bacteria the cell wall has a muerin region thicker than Gramm
negative bacteria. The individual layers of muerin are connected by peptide cross
bridges. This forms a very rigid framework for the cell wall. Teicoic acid (another
polysaccharide) is also associated with the cell wall, and we utilise this to make it
possible to identify the bacteria by immunological means.

Gram negative bacteria contain less muerin and have no teicoic acids. The cell wall is
more susceptible to mechanical damage. With these bacteria there are further layers
around the muerin consisting of lipid material. These are layers of polysaccharide, a
layer of phospholipids, and next to the muerin is a layer of phophslipid. These act as a
barrier to substances passing from the outside into the cell, such as penicillin, and also
dyes, which is why these bacteria do not take up the Gramm stain. These layers also
provide resistance to phagocytosis, therefore the bacteria are resistant to lysozymes.

The cell wall is a good target for antimicrobial drugs. Lysozymes attack the
polysacchyaride ‘backbone’ of the muerin, completely destroying the cell wall in
Gramm positive bacteria. In Gramm negative bacteria the lipid-constituent layer
remains unharmed, but the muerin layer will be destroyed. However, the bacterial cell
will remain alive.

Some bacteria can survive without a cell wall. The mycoplasma is the smallest known
bacteria to survive outside living cells, and they do not have a constant shape, i.e. they
are pleomorphic.

The capsule surrounds the cell wall, and usually is comprised of polysaccarhide, and
is loosely attached to the cell wall. There is a direct correlation between the
possession of a capsule and virulence of the organisms, e.g. Diplococcus pneumoniae
will produce pneumonia if it has it’s capsule, but without the capsule the bacteria is
less likely to produce the disease. The capsule also protects the bacteria from being
phagocytised.

Within the bacteria there is no nucleus, mitochondria, endoplastic reticulum,


lysosomes or membranous structures. There is a single strand of DNA, forming a
circular chromosome, and some ribosomes are also present.

Bacterial motility.

The usual structures to aid motility are the flagellae, which are sited on the outside of
the cell membrane. They are not found on cocci, but are on most bacilli and some
spirilla. They are usually formed from a protein material. They can be arranged in a
number of ways :

Monotrichous
Amphitrichous
Lophotrichous
Peritrichous

A pili is a structure used for adhesion found on the outside of some bacteria. They are
almost are exclusive to Gramm negative organisms, and appear much shorter than
flagellae. Genetic material can be exchanged between bacteria at the site of the pili.

Endospores.

These are round or oval bodies, formed inside the bacterial cell in adverse conditions
to help the bacteria survive. They are not reproductive structures, but a mechanism
which aids survival. Endospores may remain viable for many years through adverse
conditions, but conditions improve the endospore will germinate and produce a new
bacterial cell. Spores can be found in soil dust and air. Spores are formed mainly by
the bacilli-type organisms, particularly those of the Bacillus and Clostridium groups.
The Bacillus group are responsible for anthrax and tetanus, whilst the Clostridium
group are responsible for botulism. Bacillus stearothermophilis is a heat resistant
spore that we can use in a clinical situation. The spores are put into an autoclave, and
after the cycle is complete they are allowed to germinate. If the autoclave is working
correctly, then all spores, including this one, will be killed. If the autoclave does not
reach the correct temperature, then this spore will survive the raised temperature, and
will germinate successfully. This then indicates that the autoclave is faulty.
Bacterial growth.

Individual bacteria at maximum size will split by binary fission into two bacteria. The
maximum size of a bacteria is the size at which maximum efficiency occurs (it grows
in volume but the increase in surface area is at a much slower rate, therefore the cell
eventually becomes inefficient). The conditions required for bacterial growth are:
1. suitable nutrients
2. suitable atmosphere
3. suitable temperature
4. suitable pH
5. suitable osmotic potential
6. mosture.

Bacteria require a readily available supply of organic nutrients, in particular oxygen,


carbon, hydrogen, nitrogen, phosphorous, and sulphur. Essential minerals indlcude
calcium, iron, potassium and magnesium, together with zinc, copper, cobalt, and
magnesium.

Different atmospheres will also benefit different organisms :


• Obligate aerobes – these carry out aerobic respiration and therefore require
oxygen from the atmosphere. They cannot survive the absence of oxygen.
• Strict anaerobes – unable to grow and reproduce in the presence of oxygen,
and usually die if oxygen is present. In areas where there is no oxygen present
(i.e. devitalised tissues) these organisms can flourish, commonly producing
gangrene in areas of damaged tissue.
• Facultative anaerobes – can grow with or without the presence of oxygen.
These are sub-divided into two groups – those that respire anaerobically
whether oxygen is present or not, and those that respire anaerobically in the
absence of oxygen and aerobically if oxygen is present.
• Microaerophilic – obligate aerobes which only require a tiny amount of
oxygen. If oxygen levels are too high, they will be destroyed.

Controlling the growth of micro-organisms.

Microbial growth is controlled by physical and chemical means. Heat and autoclaves
represent the physical method, whilst disinfectants represent the chemical method.
The terms ‘sterilisation’, ‘disinfection’, ‘antisepsis’, and ‘germicide’ all refer to the
destruction of micro-organisms. Sterilisation is the process of destroying all forms of
microbial life, including spores. It is absolute, and there are no degrees of sterilisation.
Disinfection is the process of destroying vegetative pathogens, but not necessarily
endospores or viruses. Disinfectants tend to reduce or inhibit microbial growth, but
normally do not sterilise. Antisepsis refers to chemical disinfection of the skin,
mucous membranes, or other living tissues. A germicide is a chemical agent that kills
microbes rapidly, but not necessarily their spores.

The terms ‘bacteriostasis’ and ‘asepsis’ refer to the suppression of microbial growth.
Bacteriostasis is a condition in which bacterial growth and multiplication are
inhibited, but not killed. If removed, growth will resume. Asepsis is the absence of
pathogens from an object or area. Aseptic technique is designed to prevent the entry
of pathogens into the body.

Basic principles of controlling microbial growth.

These apply to all methods of control of microbial growth :


1. agent used must be able to affect the micro-organisms directly;
2. the item must be cleaned so as to remove extraneous soil;
3. moisture is essential for the action of chemical agents;
4. killing of micro-organisms is not instantaneous, and the time required depends
upon :
a. the nature of the organism
b. the nature of the agent used
c. the numbers of organisms present
d. the temperature.

Gramm positive organisms are more susceptible to disinfectants and antiseptics than
Gramm negative organisms. Pseudomonads are resistant to chemical agents, and will
actively grow in some disinfectants and antiseptics. They can also survive in simple
saline solutions, and are resistant to many antibiotics. They are a major cause of
problems in hospitals as they are opportunistic pathogens in the absence of the normal
flora of the body, i.e. when the normal flora has been suppressed during abti-biotic
therapy.

Micro-organisms are also more susceptible to chemical agents during their growth,
but with an increase in age comes an increase in resistance. Endospores are much
more resistant than vegetative cells, e.g. spores of C. Botulinum will survive after 5½
hours of boiling. Heat is more effective under acidic conditions, as are some chemical
agents.

Anti-microbial agents work by altering the permeability of the cell membrane,


causing leakage of the contents and affecting growth. Other agents work by damaging
the proteins or nucleic acids within the cell. If these proteins are destroyed then the
cell is unable to synthesise proteins, leading to cell death.

Physical methods of destruction.


Heat.
This is rapid and penetrates objects as well as clumps of micro-organisms, and all
types of organism can be destroyed. It can be used in either a dry or moist form. In it’s
dry form is kills by the oxidation effect, denaturing the cell’s proteins. In it’s moist
form because the presence of the water allows the hydrogen bonds in the proteins to
be broken down, denaturing the protein by a different method.

With moist heat spores and bacteria are killed more rapidly at a given temperature for
two reasons – chemically the proteins are denatured due to the breaking of the
hydrogen bonds, whilst physically hot water has a greater heat content than air at the
same temperature. Moist heat methods include boiling and autoclaving.

Dry heat methods include flaming, incineration, and hot air sterilisation. Flaming is
100% effective, and is often used to needles or forceps, but it has limited use
otherwise. Incineration rapidly kills micro-organisms and is reliable. The use of hot
ovens is not as efficient as moist heat, as higher temperatures are required for longer
periods of time to ensure cell death, e.g. 120oC of dry heat will take 8 hours to achieve
sterilisation. This is reduced to 20 minutes at 180oC. Sterilisation by this method is
also effective for enclosed containers, and also items made from glass or metal, but
not cloth or rubber.

Filtration.
This is the passing of a gas or liquid through a filter with pores small enough to
prevent bacteria passing through. Viruses and larger proteins can also be removed.
This method is used for liquids which would be destroyed by heat, i.e. antibiotics, and
also for air-filtration.

Radiation.
Gamma rays will penetrate cells, killing them by destroying their DNA. This method
is used especially for scalpel blade sterilisation. Ultra-violet light may also be used,
especially for vaccine sterilisation, but this works very slowly.

Chemical methods of destruction.

Very few chemical agents actually achieve sterility, but they do reduce the microbial
population to safe levels, destroying pathogens. Chemical agents should be selected
so as to kill the organisms as quickly as possible. The following table illustrates some
of the commonly used chemical agents.
Agent Action Uses.
Phenol. Disrupts the plasma No longer widely used due to its
membrane and denatures irritating properties, and may also be
proteins. teratogenic. It’s effects are also
cumulative. It is the standard for
measuring the effectiveness of other
disinfectants (phenol co-efficient).
Phenolics Disrupts the plasma These are derivatives of phenol and
membrane and denatures are reactive even in the presence of
proteins. organic material. Used to disinfect
surfaces and instruments as well as
skin surfaces and mucous membranes.
Examples include cresol, Lysol,
thymol, xylenol.
Halogens. Iodine – inhibits protein These agents may act alone or as a
function and is a strong component of inorganic/organic
oxidiser compounds. They are both effective
Chlorine – forms antiseptics.
hypochlorous acid, a strong
oxidiser which alters cellular
components.
Alcohols. Dehydrates and is a lipid Bacvtericidal and fungicidal, but
solvent, acting on the cell ineffective against endospores and
membrane. non-enveloped viruses. Used for skin
swabbing and also wiping instruments.
Heavy Denature proteins Germicidal and antiseptic in action.
metals and
their
compounds.
Dyes. Interfere with cellular Antiseptic and antifungal.
oxidation.
Quarternary Inhibit enzymes, denature Surface-active agents. Bactericidal,
ammonium proteins and disrupt plasma bacteriostatic, fungicidal, and
compounds membranes. virucidal against enveloped viruses.
(QAC) They are colourless, odourless,
tasteless, non-toxic, stable and easily
diluted. Used for skin antisepsis and
washing/disinfecting instruments,
utensils, and rubber goods.
Acids and Cause hydrolysis and Used on the skin to control
alkalis. denaturing of proteins. dermatophytes, and as food
preservatives to inhibit mould
growths.
Aldehydes. Inactivate proteins. Very effective agents. Used to
preserve and embalm specimens. Will
also inactivate micro-organisms and
vaccines. Common example id
formalin.
Oxidising Oxidise cellular components Ozone can be used to replace chlorine
agents. of cells. for disinfecting water. Hydrogen
peroxide is used as an antiseptic in
deep wounds.

The normal flora of the body.

Many organisms inhabit the body without causing us any harm, and is some cases are
positively beneficial and essential for health. Mutuals are bacteria which actually aid
some process in the body, such as vitamin K-producing bacteria in the gut.
Commensals benefit from living on the body, but we gain no benefit directly from
their presence. These mostly live on the skin. The areas on the body which mainly
have normal flora are those areas which have an exterior connection, i.e. mouth.
Those that have no exterior connection have no flora.

The normal flora will utilise any nutrients that are available, preventing their use by
pathogens. They also maintain certain physiological conditions within the body, such
as pH, making it impossible for pathogens to flourish. They can also produce
secretions which inhibit invading micro-organisms. Flora on the epithelial layers
occupy specific attachment sites, and once these attachment sites are occuplied there
is no-where for pathogens to attach and establish themselves.

The major constituent of the normal flora is S. albus, making up around 90% of the
population. Another common organism is S. aureus, found commonly in the nose and
anus. Diphtheroids occur cutaneously, and commonly inhabit oily areas such as the
sebaceous glands. Streptococci are seldom found in normal flora, but α-haemolytic
streptococci exist in the mouth and can spread to the skin. Gram negative bacilli form
a very small proportion of the flora, and tend to occur in moist areas only as they
cannot survive in dry areas.

Nail flora – this is generally similar to that of skin, but in addition particles of dust
and other materials can be trapped which contain spores and bacteria, eg, aspergillus,
penicillium.

Oral and upper respiratory tract – pharynx and trachea contain many potentially
pathogenic organisms, and this is often the site of the initial colonisation of
pathogens.

Intestinal tract – the concentration of organisms increase towards the end of the small
intestine. There is a high concentration in the colon, a high percentage of which are
anaerobic (more than 90%).

Urogenital tract – depends upon age, pH, and hormone levels.

There are certain conditions in which normal flora may cause infection :
• when taking antibiotics that are not specific to one type of bacteria, thereby
killing all bacteria. This destroys the normal flora and the other organisms,
such as fungi and yeasts, will multiply, causing infection, e.g. thrush.
• Extreme and prolonged fatigue .
• Debilitated patients, especially those recovering from major surgery, cancer,
diabetes or chest conditions.
• The use of drugs such as steroids, and possible drugs of abuse.
• Large amounts of alcohol.
• Mechanical damage to the skin or tissues.
• Transmission of normal flora to another region.

Microbial invasiveness and disease production.

Disease production depends upon a number of factors :


1. host/microbial factors –
a. portal on entry – the organisms enters the wrong portal it cannot cause
disease.
b. actual species – only certain micro-organisms cause disease.
c. numbers of micro-organisms – salmonella poisoning requires over 1
million organisms, whereas only 1 rickittsia will cause Scrub Typhus.
d. ability to multiply.
e. must attach to epithelial surfaces.
f. needs correct metabolic and nutritional situation.

2. specific microbial factors –


a. production of a capsule for protection
b. toxin production – endotoxins are part of the bacterial cell wall which
is released on lysis or destruction. They are associated with Gramm
negative bacteria and tend to be non-specific. They result in headache,
fever, and nausea. Exotoxins are secreted into the surrounding
environment and are very powerful. They are mainly associated with
Gramm positive bacteria, and it is the exotoxin which causes the
disease, e.g. clostridium tetani produces a toxin which results in
Lockjaw.
c. haemolysins – released by bacteria to break down red blood cells
d. leukocidins – released by bacteria to destroy leukocytes
e. coagulase – produced by some staphylococci to promote blood clotting
f. fibrinolysis – produced by streptococci and breaks down a blood clot
by destroying the fibrin
g. hyaluronidase – breaks down hyaluronic acid, enabling bacteria to get
in between the host’s cells
h. collagenase – breaks down collagen in connective tissues
i. penicillinase – breaks down penicillin and other similar substances.

Summary of factors necessary for disease to occur :


The correct species must enter the right portal in sufficient numbers for that
species to cause disease. They must have the ability to attach to epithelial
surfaces, have the correct nutrients, temperature and environmental conditions
for growth, and produce substances such as capsules, toxins and enzymes
necessary for their survival and growth.

States of bacterial infection.

State of infection Description Examples


Subclinical (apparent) No detectable clinical Asymptomatic gonorrhea
symptoms of infection in women and men
Dormant (latent) Carrier state Typhoid carrier
Accidental Zoonosis and Anthrax
environmental exposure
Opportunistic Infection caused by normal Candida infection
flora or transient bacteria
when normal host defences
compromised
Primary Clinically apparent Shigella, dystentery
invasion and
multiplication of microbes
in body tissues causing
local tissue injury
Secondary Microbial invasion Bacterial pneumonia, viral
subsequent to primary lung infections
infection
Mixed Two or more microbes Anaerobic abscess
infecting the same tissue
Acute Rapid onset, brief duration Diphtheria
Chronic Prolonged duration Tuberculosis, leprosy
Localised Confined to a small area or Staphylococcal boil
an organ
Generalised Disseminated to many Gramm negative
body regions bacteraemia
Pyogenic Pus forming Streptococcal infection
Retrograde Microbes ascending a duct E.coli urinary tract
or tube against the flow of infection
secretions or excretions
Fulminant Infections that occur Pneumonic plague
suddenly and intensely