Bacterial Agents
Bacterial cell structure
Bacteria are single celled organisms
which typically range in size 0.3µm to 14µm (1µm = 1/1000 of a
millimetre). Whilst exceedingly small they are fully functioning
organisms, obtaining their nutrition from their environment, and able to
manufacture the complex substances necessary for life and reproduction.
The cell structure is simpler than that of other organisms as there is
no nucleus nor are there membrane bound organelles. Instead their
control centre containing the genetic information is contained in a
single loop of DNA. Some bacteria have an extra circle of genetic
material called a plasmid. The plasmid often contains genes that give
the bacterium some advantage over other bacteria. For example it may
contain a gene that makes the bacterium resistant to a certain
antibiotic.
Bacteria vary enormously in the substances which they can utilise as
food and the environments they can survive in, from high altitude to the
bottom of the deepest seas, to hot sulfur springs and even storage pools
for nuclear waste. Whilst some organisms can survive in a wide range of
situations, others are extremely specific.
Microbiologists classify bacteria by a variety of means including:
shape, motility, reactions to various stains, whether or not they need
oxygen to survive, their ability to utilise different energy sources,
their appearance when grown in the laboratory, whether or not they
ferment certain sugars and the temperatures at which they live. The cell
diagram at the top of the page shows the major features of a bacterial
cell, however not all bacteria show all feature.
Bacterial
morphology
Bacteria
are, in part, classified into groups according to their basic shapes or
morphology: spherical (cocci), rod (bacilli), spiral (spirilla), comma
(vibrios) or corkscrew (spirochaetes). They can exist as single cells,
in pairs, chains or clusters.
Staining
methods are also used in identifying bacteria. The best known staining
methods for bacteria are Gram's stain and Ziehl-Neelsen acid-fast
staining. Other common methods include Albert's, Giemsa, negative and
silver stains
Gram's stain
Gram's stain divides all bacteria into
one of two groups, gram positive and gram negative. There are numerous
variations of the Gram staining method, in fact it was the subject of
the first research project I conducted back in 1966. Fundamentally a
film of bacteria is spread on a microscope slide and then heat fixed by
passing through a flame three or four times. The method may also be used
on histological sections, with some minor variations of technique. The
film, is then flooded with a solution of crystal violet for a few
seconds. Next the slide is washed and then flooded with dilute iodine
solution. Following this an attempt is made to remove the stain using
70% ethanol (some methods use acetone or an acetone/ethanol mixture),
this only lasts a few seconds, it being relatively easy to decolourise
bacteria if the solvent is used for two long a period, particularly if a
more aggressive solven is used. The smear is then counter-stained with a
red or yellow stain. Bacteria that retain the crystal
violet/iodine stain and appear blue/black are said to be Gram positive.
Ziehl
Neelsen stain
Some bacteria are acid resistant, in
particular members of the Mycobateria. In acid fast staining bacteria
are differentiated according to whether or not, after staining, they can
be decolourised by sulfuric or hydrochloric acid, the best known of
these staining methods is Ziehl-Neelsen. A heat killed smear or
histological section is first stained with carbol-fuchsin (a solution of
the red dye fuchsin in a solution of phenol. The slide is then washed in
water, and an attempt to remove the stain is made using either dilute
hydrochloric or sulfuric acid or acidified alcohol (3-5% hydrochloric
acid in ethanol is commonly used). The preparation is then
counter-stained with either a blue or green stain, typically methylene
blue. Acid-fast organisms show up as red.
Other staining techniques
There are
numerous other specialised staining techniques, develioped to show the
presence or absence of spores, the presence or absence of flagellae etc.
Students are advised to consult either one of the sites listed below or
a textbook of bacteriological technique:
Potential Bacterial agents
The letters in (brackets) represent the
NATO military codename(s). The letters in [square brackets] represent
the National Center for Emerging and Zoonotic Infectious Diseases
(NCEZID) classification of organisms.
Bacillus anthracis causes anthrax and is one of the most
deadly agents to have been used as a biological weapon. It is classified
by the US Centers for Disease Control and Prevention (CDC) as a Category
A agent, posing a significant risk to national security. The
gram-positive, rod-shaped anthrax spores are found naturally in soil,
can be easily cultured in a laboratory, and last for many years, as
spores, in the environment. Anthrax has been used as a biological weapon
for about a century. Letters containing powdered anthrax spores were
intentionally mailed through the US postal system in 2001 affecting 22
people of which five died. Experiments with the use of anthrax have been
undertaken by all countries attempting to use biological weapons.
Brucella abortus causes
contagious abortion in ruminants and brucellosis in humans. B.
abortus is a Gram-negative rod shaped bacteria that is
non-motile, nor does it create capsule slime. It does produce
endospores, which enable survival under long-term starvation and
dessication. This heterotrophic bacterium carries out either aerobic or
anaerobic respiration. This bacterium, as an intracellular pathogen,
enters phagocytes, such as macrophages, in humans and in cows. It
attaches to the endoplasmic reticulum of these cells. In humans
brucellosis has both an acute and a chronic phase. The chronic phase
will last as long as the host is alive without treatment. Acute symptoms
include fever, chills, headache, backache, weakness, and weight loss.
The chronic symptoms are usually recurring joint pain, fatigue, and
headaches. There is an antibiotic regimen for humans who come in contact
with the disease that includes the antibiotics rifampin and doxycycline
together.
Brucella melitensis is a
Gram-negative coccobacillus bacterium from the Brucellaceae
family. The bacterium causes ovine brucellosis. It affects primarily
sheep and goats, but cases have also been observed in cattle, yaks,
water buffalo, camels, alpacas, dogs, horses and pigs. Humans can
become infected. B. melitensis is the most pathogenic of
the brucellae, for humans.
Brucella suis, the cause of
porcine brucellosis, can also infect humans. B. suis is a
Gram-negative, facultative, intracellular coccobacillus, capable of
growing and reproducing inside of host cells, specifically phagocytic
cells. They are not spore-forming, capsulated, or motile. B. suis
was the first biological agent weaponized in 1952 in the USA, and was
field-tested with B. suis-filled bombs called M33 cluster bombs. It is,
however, considered to be one of the agents of lesser threat because
many infections are asymptomatic and the mortality is low, but it is
used more as an incapacitating agent.
Burkholderia mallei is the
cause of glanders,
a zoonotic disease that primarily affects horses, donkeys, and mules,
but can also be seen in other animals e.g. goats, dogs, and cats.
Glanders is rare in humans. B. mallei is a Gram-negative,
coccobacillius it is non-motile and aerobic. During World War I, the
Germans used B. mallei to infect animals that were being sent
from neutral countries to the Allies with glanders.
Burkholderia pseudomallei the
cause of melioidosis
in humans also known as Whitmore's disease. B. pseudomallei
is practically identical to B. mallei (they share about 99% of
their DNA), to the extent that it is very difficult to tell them apart,
and some authorities regard them as different strains of the same
organism. B. pseudomallei is a Gram-negative, coccobacillius,
it is non-motile and aerobic. There are several types of melioidosis
infection, each with their own set of symptoms. It is important to note
that melioidosis has a wide range of signs and symptoms that can be
mistaken for other diseases such as tuberculosis or more common forms of
pneumonia.
Chlamydophila psittaci
is a lethal intracellular bacterial species that may cause endemic avian
chlamydiosis, epizootic outbreaks in mammals, and respiratory
psittacosis in humans. C. psittaci undergoes several
transformations during its lifecycle it exists as an elementary
body (EB) between hosts The EB is not biologically active, but is
resistant to environmental stresses and can survive outside a host. The
lifecycle of C. psittaci is divided between the elementary
body which is able to infect new hosts, but cannot replicate, and the
reticulate body, which replicates, but is not able to cause new
infection.
Clostridium botulinum is a
Gram-positive, rod-shaped, spore-forming bacterium, its toxins cause
botulism. It is an obligate anaerobe, meaning that oxygen is poisonous
to the cells. However, C. botulinum tolerates traces of oxygen
due to the enzyme superoxide dismutase, which is an important
antioxidant defense in nearly all cells exposed to oxygen. C.
botulinum forms protective spores when conditions for survival
are poor. The spore has a hard protective coating that encases the key
parts of the bacterium and has layers of protective membranes. Within
these membranes and the hard coating, the dormant bacterium is able to
survive for years. C. botulinum produces seven neurotoxins
(types A-G). These are the most potent toxins known (as little as 30 ng
is sufficient to cause illness and possibly death), and are responsible
for botulism, a severe and often fatal neuroparalytic intoxication. Two
related organisms Clostridium butyricum and Clostridium
baratii may also produce the same toxins.
Corynebacterium diphtheriae
the cause of diphtheria, is a nonmotile, aerobic, noncapsulated,
club-shaped, Gram-positive rod-shaped bacterium. Some strains produce a
potent exotoxin. C. diphtheriae is only able to produce its
exotoxin when the bacterium is infected by a bacteriophage which
provides it with the toxin-producing gene. The symptoms of
diphtheria include pharyngitis, fever, swelling of the neck or area
surrounding the skin lesion. Diphtheritic lesions are covered by a
pseudomembrane. The toxin is distributed to distant organs by the
circulatory system and may cause paralysis and congestive heart failure.
Coxiella burnetii is an
obligate intracellular bacterial pathogen, and is the causative agent of
Q fever. C. burnetii is a small Gram-negative, cocco-bacillary
bacterium that is highly resistant to environmental stresses such as
high temperature, osmotic pressure, most disinfectants and ultraviolet
light. C. burnetii was originally classified as a
rickettsia, but when differences from other rickettsia became apparent
it was assigned its own genus. C. burnetii is one of the
most infectious organisms known, only 1-10 organisms are needed to
infect 50% of the population. C. burnetii is one of
seven biological organisms that were weaponised by the USA during their
biological warfare programme.
Escherichia coli is a
Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of
the genus Escherichia that is commonly found in the lower
intestine of warm-blooded organisms. Most E. coli strains are
harmless, but virulent strains can cause gastroenteritis, urinary tract
infections, neonatal meningitis, hemorrhagic colitis, and and has been
implicated in Crohn's disease. The harmless strains are part of the
normal microbiota of the gut, and can benefit their hosts by producing
vitamin K2, and preventing colonisation of the intestine with pathogenic
bacteria. Under favorable conditions, it takes as little as 20 minutes
to reproduce.
Francisella tularensis is
the cause of tuluraemia, also known as Pahvant Valley plague, rabbit
fever, deer fly fever, and Ohara's fever. Tularaemia is a bacterial
zoonotic disease of the northern hemisphere. F. tularensis is
an aerobic Gram-negative coccobacillus. It is a non-sporing and
non-motile organism. In nature, the disease may be spread in a number of
ways, person to person by droplet infection, by arthropod vector, or
contact with infected animals, or ingestion of contaminated water or
food. The Soviet Red Army used F. tularensis against German
troops in the battle of Stalingrad during World War II.
Listeria monocytogenes is a
facultative anaerobic, Gram positive, rod-shaped, motile,
non-spore forming bacterium. It can grow and reproduce inside the host's
cells and is one of the most virulent food-borne pathogens: 20 to 30% of
foodborne listeriosis infections in high-risk individuals may be fatal.
L. monocytogenes is able to reproduce at 0°C. Invasive
infection by L. monocytogenes causes the disease listeriosis.
When the infection is not invasive, any illness as a consequence of
infection is termed febrile gastroenteritis. The manifestations of
listeriosis include sepsis, meningitis (or meningoencephalitis),
encephalitis, corneal ulcer, pneumonia, and intrauterine or cervical
infections in pregnant women, which may result in spontaneous abortion
in the second to third trimester or stillbirth.
Mycobacterium tuberculosis
the cause of tuberculosis has an unusual, waxy coating on its cell
surface primarily due to the presence of mycolic acid. This coating
makes the cells impervious to Gram staining, and as a result, M.
tuberculosis can appear either Gram-negative or Gram-positive.
Acid-fast stains such as Ziehl-Neelsen, or fluorescent stains such as
auramine are used instead to identify M. tuberculosis
microscopically. M. tuberculosis is rod-shaped, a strict
aerobe, is non-sporing, and is non-motile. M. tuberculosis is
unusually slow growing, reproducing by binary fission every 18-24 hours.
Primarily a pathogen of the mammalian respiratory system, it normally
infects the lungs. M. tuberculosis is a member of a group of
closely related organisms comprising: M. tuberculosis, M.
paratuberculosis, M. africanum, M. orygis. M.
bovis and the Bacillus Calmette-Guérin strain, M. microti,
M. canetti, M. caprae, M. pinnipedii, M.
suricattae, and M. mungi. M. tuberculosis is
an unlikely BW agent because of its slow growth, and the fact that the
majority of the population have been immunised.
Rickettsia prowazekii is a
small, Gram-negative, obligately intracellular, rod-shaped
bacterium. R. prowazekii is the causative agent of
epidemic typhus, also called louse-borne typhus. Epidemic typhus is
spread to people through contact with infected body lice. Symptoms of
epidemic typhus begin within 2 weeks after contact with infected body
lice. Signs and symptoms may include: fever and chills, headache, rapid
breathing, body and muscle aches, rash, cough, nausea, vomiting, and
confusion. Some people can remain infected, without symptoms, for years
after they first get sick.
Rickettsia rickettsii the
cause of Rocky Mountain spotted fever, is a Gram-negative,
intracellular, coccobacillus bacterium that is around 0.8 to 2.0 μm
long. R. rickettsii is one of the most pathogenic of
Rickettsia. The most common cause of infection is bites by infected
ticks. The most common hosts for R. rickettsii are
ticks in the family Ixodidae. Typical symptoms of RMSF
can appear 2 - 14 days after exposure and include fever, headache,
depression, nausea, vomiting, and a skin rash called purpura or
petechiae. Sometimes the rash occurs 2 to 5 days after the onset of the
fever. Serious cases of RMSF can include central nervous system,
pulmonary, or hepatic injuries.
Rickettsia typhi is a small,
Gram-negative, rod-shaped, obligately intracellular bacterium. R.
typhi is the cause of murine or endemic typhus. R. typhi is
transmitted primarily by the rat flea, Xenopsylla cheopis,
although lice and mites are also potential vectors. Rodents (mainly Rattus
norvegicus, and Rattus rattus) are considered the main
reservoir of bacteria, but other vertebrate hosts may serve as reservoir
including house mice, shrews, opossums, skunks, and cats. Murine typhus
symptoms are similar to those of epidemic typhus, although the former is
usually less severe. The incubation period is usually more prolonged
than that of epidemic typhus. Symptoms include headache, arthralgia and
ill feeling, with or without a low grade fever. Onset is characterized
by persistent headache, a high grade fever, and a cutaneous rash
predominating on the trunk. The rash is usually less apparent than in
epidemic typhus, and occasionally absent.
Salmonella
enterica are rod-shaped, motile, facultative aerobic,
Gram-negative bacteria. S. enterica contains a large number of serovars
or serotypes, which can infect a broad range of vertebrate hosts. The
individual members range from being highly host-adapted to those
displaying a broad host range The most significant serovars as far as
human disease is concerned are: Enteritidis, Hadar,
Heidelberg, Infantis, Paratyphi, Typhi, and Typhimurium. In
fact there are more than 2,500 serovars. Of these the last three
are considered to be potential BW agents.
- S. enterica Paratyphi,
there are three varieties of S. enterica Paratyphi, A, B, & C.
They cause paratyphoid, a potentially severe and occasionally
life-threatening bacteraemic illness. While fever and
gastrointestinal symptoms are common, the clinical presentation
varies, including mild and atypical infections. Infections caused by
Salmonella enterica serotypes Paratyphi A, B (tartrate negative),
and C are often characterized by insidious onset of sustained fever,
headache, malaise, anorexia, relative bradycardia, constipation or
diarrhea, and non-productive cough. However, mild and atypical
infections may occur.
- S. enterica Typhi, is
the cause of typhoid fever a systemic infection, usually through
ingestion of contaminated food or water. The acute illness is
characterized by prolonged fever, headache, nausea, loss of
appetite, and constipation or sometimes diarrhoea. Symptoms are
often non-specific and clinically non-distinguishable from other
febrile illnesses. However, clinical severity varies and severe
cases may lead to serious complications or even death.
- S. enterica Typhimurium,
is the cause of paratyphoid fever a life-threatening
illness. The acute illness is characterised by a sustained fever
that can be as high as 39 - 40°C, weakness, abdominal pain,
headache, diarrhea or constipation, cough, loss of appetite. Some
people with paratyphoid fever develop a rash of flat,
rose-colored spots.
Shigella spp. cause the
infectious, intestinal human disease shigellosis, they are non-spore
forming, non-motile, rod-shaped Gram-negative bacteria. Most who are
infected with Shigella develop diarrhea, fever, and stomach cramps
starting a day or two after they are exposed to the bacteria.
Shigellosis usually resolves in 5 to 7 days. Some people who are
infected may have no symptoms at all, but may still pass the Shigella
bacteria to others. S. dysenteriae serogroup A causes
deadly epidemics mainly in developing countries, S. boydii
serogroup C is restricted to the Indian subcontinent, and S.
flexneri serogroup B and S. sonnei serogroup D are prevalent in
developing and developed countries, respectively. S. flexneri is
also responsible for the worldwide endemic form of shigellosis. Members
of the genus are highly infectious, and cause 1 million deaths annually
worldwide.
Vibrio cholerae - the cause
of cholera, is a Gram-negative, comma-shaped, halophilic, a
facultatively anaerobic, highly motile bacterium. Cholera is a diarrheal
disease, easily mistakable for several others; however, there are some
clinical features that are characteristic and can help make the
diagnosis. The presence of watery diarrhea with the appearance of rice
wter is characteristic. This is even more impressive when associated
with acute severe dehydration. Other symptoms may include: abdominal
pain, vomiting, cramping rectal pain and other cramps, abnormally slight
or infrequent urination, dry mucosae, fever is less common, mental
status alteration, from alert to restless, somnolent and even comatose
Yersinia pestis (formerly
known as Pasteurella pestis) the cause of plague
is a gram-negative, non-motile, non-sporing, rod-shaped, facultatively
anaerobic, coccobacillus bacterium. Symptoms of plague include fever,
weakness and headache. Usually this begins one to seven days after
exposure. There are three forms of the disease. In the bubonic form
there is also swelling of lymph nodes, while in the septicemic form
tissues may turn black and die, and in the pneumonic form shortness of
breath, cough and chest pain may occur. Bubonic and septicemic plague
are generally spread by flea bites or handling an infected animal. The
pneumonic form is generally spread person to person through the air via
infectious droplets.
- Bubonic plague - When an
infected flea bites a human and contaminates the wound with
regurgitated blood, the plague-causing bacteria are passed into the
tissue. Y. pestis can reproduce inside cells, so even if
phagocytosed, they can still survive. Once in the body, the bacteria
can enter the lymphatic system. Y. pestis spreads through the
lymphatic vessels of the infected person until it reaches a lymph
node, where it causes acute lymphadenitis. The swollen lymph
nodes form the characteristic buboes associated with the disease. If
the lymph node is overwhelmed, the infection can pass into the
bloodstream, causing secondary septicemic plague and if the lungs
are seeded, it can cause secondary pneumonic plague.
- Septicemic plague - The
lymphatic system ultimately drains into the bloodstream, so the
plague bacteria may enter the blood and travel to almost any part of
the body. In septicemic plague, bacterial endotoxins cause
disseminated intravascular coagulation (DIC), causing tiny clots
throughout the body and possibly ischemic necrosis (tissue death due
to lack of circulation) from the clots. DIC results in depletion of
the body's clotting resources, so that it can no longer control
bleeding. Consequently, there is bleeding into the skin and other
organs, which can cause red and/or black patchy rash and haemoptysis
an haematemesis. There are bumps on the skin that look somewhat like
insect bites; these are usually red, and sometimes white in the
center. Untreated, septicemic plague is usually fatal. People who
die from this form of plague often die on the same day symptoms
first appear.
- Pneumonic plague - The
pneumonic form of plague arises from infection of the lungs. It
causes coughing and thereby produces airborne droplets that contain
bacterial cells and are likely to infect anyone inhaling them. The
incubation period for pneumonic plague is short, usually two to four
days, but sometimes just a few hours. The initial signs are
indistinguishable from several other respiratory illnesses; they
include headache, weakness and spitting or vomiting of blood. The
course of the disease is rapid; unless diagnosed and treated soon
enough, typically within a few hours, death may follow in one to six
days; in untreated cases mortality is nearly 100%.