Difference between Gram -ve and Gram +ve Bacteria

Bacteria are generally classified into two groups on the basis of Gram staining based on the amount of peptidoglycan present in the cell wall.Though it is found that Gram positive and gram negative bacteria have certain other variable characteristics other than merely the result of Gram staining: Gram positive- 1.They retain the crystal violet stain after gram staining. 2.They have peptidoglycan layer thickness about 20-80nm and about 40-60 layers thick. 3.Peptidoglycan is much more mesh like and has interpeptide cross linkages. 4.cell wall has techoic acids which confer negative charges to the cell wall. 5.Have only one periplasmic space. Gram negative- 1.They retain the colour of safranine,the counter stain of gram stain, and not the colour of the primary stain crystal violet. 2.The cell wall has an additional layer outside the peptidoglycan layer known as the outer membrane. 3.The outer membrane contains lipid-A,core polysaccharide and the O-side chain. 4.Lipid A portion of core polysaccharide is known as endotoxin and help in virulence. 5.O side chain is also known as o specific antigen as it elicits an immune response. 6.Peptidoglycan has a thickness of 2-7 nm . 7.It has two periplasmic spaces on either side of petidoglycan layer.

Clssification of Bacteria

General Structure of Bacterium

The structural difference between the Prokaryotes and the Eukaryotes are so significant that some biologist think that these two groups merit the status of Super Kingdoms. The main distinguishing feature between the Prokaryotes and Eukaryotes is the lack of a true nucleus in the former.

The general size of a prokaryotic cell is about 1-2 um. Note the absence of membrane bound organelles There is no true nucleus with a nuclear membrane The ribosome's are smaller than eukaryotic cells The slime capsule is used as a means of attachment to a surface Only flagellate bacteria have the flagellum Plasmids are very small circular pieces of DNA that maybe transferred from one bacteria to another.

1.2.2 Function of Prokaryotic cell structures

Structure

Function

Cell Wall

Made of murein (not cellulose), which is a glycoprotein or peptidoglycan (i.e. a protein/carbohydrate complex). There are two kinds of bacterial cell wall, which are identified by the Gram Stain technique when observed under the microscope. Gram positive bacteria stain purple, while Gram negative bacteria stain pink. The technique is still used today to identify and classify bacteria. We now know that the different staining is due to two types of cell wall

Plasma membrane

Controls the entry and exit of substances, pumping some of them in by active transport .

Mesosome

A tightly-folded region of the cell membrane containing all the membrane-bound proteins required for respiration and photosynthesis. Can also be associated with the nucleoid. This is now thought to be an artifact of the electron microscope and not a real structure.

Cytoplasm

Contains all the enzymes needed for all metabolic reactions, since there are no organelles

Ribosome's

The smaller (70 S) type are all free in the cytoplasm, not attached to membranes (like RER). They are used in protein synthesis which is part of gene expression.

Naked DNA

Nucleoid is the region of the cytoplasm that contains DNA. It is not surrounded by a nuclear membrane. DNA is always circular (i.e. a closed loop), and not associated with any proteins to form chromatin. Sometimes confusingly referred to as the bacterial chromosome

Slime Capsule

A thick polysaccharide layer outside of the cell wall, like the glycocalyx of eukaryotes. Used for sticking cells together, as a food reserve, as protection against desiccation and chemicals, and as protection against phagocytosis. In some species the capsules of many cells in a colony fuse together forming a mass of sticky cells called a biofilm. Dental plaque is an example of a biofilm.

Bacteria of Medical Importance

Bacteria That Cause Human Disease

Only a small fraction of the thousands of species of bacteria on the earth cause disease in humans. Bacterial infection can be prevented by killing bacteria with heat, as in sterilization and pasteurization. If a bacterial infection does occur, doctors may treat it with antibiotics. However, overuse of antibiotics in recent years has enabled the development of strains of bacteria that are resistant to antibiotics, such as Mycobacterium tuberculosis, which causes tuberculosis.

Bacterium

Disease

Bacillus
Bacillus anthracis	Anthrax
Bacillus cereus	B. cereus food poisoning
Clostridium botulinum	Botulism
Clostridium perfringens	Clostridial myonecrosis (gas gangrene)
Clostridium tetani	Tetanus (lockjaw)
Corynebacterium diphtheriae	Diphtheria
Escherichia coli	Diarrhea
Klebsiella pneumoniae	Bronchopneumonia
Legionella pneumophila	Legionnaire's disease
Mycobacterium leprae	Leprosy
Mycobacterium tuberculosis	Tuberculosis
Salmonella species	Salmonella
Salmonella typhi	Typhoid fever
Salmonella typhimurium	Salmonella gastroenteritis
Shigella dysenteriae	Bacillary dysentery
Shigella species	Shigellosis
Yersinia enterocolitica	Yersiniosis, gastroenteritis
Yersinia pestis	Plague
Yersinia pseudotuberculosis	Mesenteric lymphadenitis
Chlamydia
Chlamydia trachomatis	Trachoma, urethritis, cervicitis, conjunctivitis
Coccobacillus
Bordetella pertussis	Pertussis (whooping cough)
Brucella species	Undulant fever
Hemophilus influenzae	Meningitis, bacterial pneumonia
Hemophilus pertussis	Pertussis (whooping cough)
Coccus
Neisseria gonorrhoeae	Gonorrhea, pelvic inflammatory disease
Neisseria meningitidis	Meningitis
Staphylococcus aureus	Pneumonia, toxic shock syndrome, skin infections, meningitis
Streptococcus pneumoniae	Pneumonia, ear infections, meningitis
Streptococcus pyogenes	Strep throat, rheumatic fever
Streptococcus species	Scarlet fever, puerperal fever
Listeria
Listeria monocytogenes	Listeriosis, perinatal septicemia, meningitis, encephalitis, intrauterine infections
Mycoplasma
Mycoplasma pneumoniae	Pneumonia
Rickettsia
Rickettsia prowazekii	Epidemic typhus, Brill-Zinsser disease (spread by lice)
Rickettsia rickettsii	Rocky Mountain spotted fever (spread by ticks)
Rickettsia typhi	Endemic typhus (murine typhus, spread by rat fleas)
Spirillus
Campylobacter fetus jejuni	Campylobacteriosis (bacterial diarrheal illness)
Spirillum minor	Rat-bite fever
Spirochete
Treponema pallidum	Syphilis
Vibrio
Aeromonas hydrophila	Gastroenteritis, septicemia, cellulitis, wound infections, urinary tract infections
Plesiomonas shigelloides	Gastroenteritis, diarrhea
Vibrio cholerae 01	Epidemic cholera
Vibrio cholerae non-01	Gastroenteritis
Vibrio parahemolyticus	V. parahemolyticus-associated gastroenteritis
Vibrio vulnificus	Wound infections, gastroenteritis, primary septicemia

Binary Fission

A method of asexual reproduction that is employed by most prokaryotes. In binary fission, the living cell divides into two equal, or nearly equal, parts. It begins when the DNA of the cell is replicated. Each circular strand of DNA then attaches to the plasma membrane. The cell elongates, causing the two chromosomes to separate. The plasma membrane then invaginates (grows inward) and splits the cell into two daughter cells through a process called cytokinesis.

Binary fission theoretically results in two identical cells. However, the DNA of bacteria has a relatively high mutation rate. This rapid rate of genetic change is what makes bacteria capable of developing resistance to antibiotics and helps them exploit invasion into a wide range of environments.

Similar to more complex organisms, bacteria also have mechanisms for exchanging genetic material. Although not equivalent to sexual reproduction, the end result is that a bacterium contains a combination of traits from two different parental cells. Three different modes of exchange have thus far been identified in bacteria.

Conjunction involves the direct joining of two bacteria, which allows their circular DNAs to undergo recombination. Bacteria can also undergo transformation by absorbing remnants of DNA from dead bacteria and integrating these fragments into their own DNA. Lastly, bacteria can exchange genetic material through a process called transduction, in which genes are transported into and out of the cell by bacterial viruses, called bacteriophages, or by plasmids, an autonomous self-replicating extrachromosomal circular DNA.

Bacteria-HOST-PATHOGEN Relationships man

Any microorganism that is able to infect a host and produce disease is a pathogen. Bacteria can cause a multitude of different infections ranging in severity from inapparent to fulminating. The capacity of
the organism to cause disease reflects its relative pathogenicity. On this basis, bacteria can be
grouped into three major groups;

a) Frank / Primary Pathogens
These are those bacteria that when isolated from patients are
considered to be the agents of the disease. They always cause disease when they infect a host
e.g. Neisseria gonorrhoea, Yersinia pestis
b) Opportunistic pathogens
These rarely if ever cause disease in immunocompetent hosts but
can cause serious infection in immunocompromised hosts. Usually, they are members of the body's normal flora that take the opportunity offered by the reduced host defences to cause
disease e.g. Staphylococcus epidermidis, Pseudomonas aeroginosa, Mycobacterium avium
c) Non Pathogens
These rarely or never cause human disease. This categorization of non pathogens may change because of adaptability of bacteria and detrimental effects on resistance mechanisms of host e.g. Archaea, Lactobacillus acidophilus

Normal Flora
Generally, opportunistic and non pathogenic bacteria are found in living tissues as normal flora.
Normal flora (normal microbiota, commensals, indigenous organisms) refers to bacteria which do not normally cause diseases and can be found commonly in healthy individuals.
They may cause diseases
(1) in an immunocompromised host,
(2) after being introduced into different body sites,
(3) overgrow in favorable conditions.
A human first becomes colonized by a normal flora at the moment of birth and passage through the birth canal. In utero, the fetus is sterile, but when the mother's water breaks and the birth process begins, so does colonization of the body surfaces. Handling and feeding of the infant after birth leads to establishment of a stable normal flora on the skin, oral cavity and intestinal tract in about 48 hours. In a healthy animal, the internal tissues, e.g. blood, brain, muscle, etc., are normally free of microorganisms. However, the surface tissues, i.e., skin and mucous membranes, are constantly in contact with environmental organisms and become readily colonized by various microbial species that form the normal flora of these membranes.

Bacterial-Host-Parasite Relationships in man

Bacteria are consistently associated with the body surfaces of animals. There are many more bacterial cells on the surface of a human (including the gastrointestinal tract) than there are human cells that make up the animal. The bacteria and other microbes that are consistently associated with an animal are called the normal flora, or more properly the "indigenous microbiota", of the animal. These bacteria have a full range of symbiotic interactions with their animal hosts. There are at least three types of relationships based on the quality of the relationship for each member of the symbiotic association.

Types of Symbiotic Associations
a) Mutualism
Refers to a symbiotic relationship in which both members in the association
benefit. For humans, one classic mutualistic association is that of the the lactic acid bacteria that live on the vaginal epithelium of a woman. The bacteria are provided habitat with a constant temperature and supply of nutrients (glycogen) in exchange for the production of lactic acid, which protects the vagina from colonization and disease caused by yeast and other potentially harmful microbes.

b) Commensalism
There is no apparent benefit or harm to either member of the
association. A problem with commensal relationships is that if you look at one long enough and hard enough, you often discover that at least one member is being helped or harmed during the association. E.g. Staphylococcus epidermidis a common inhabitant of
the human skin

c) Parasitism
Refers to a relationship in which one organism called parasite grows, feeds
and is sheltered on or in a different organism called a host while contributing nothing to the survival of its host. In microbiology, the mode of existence of a parasite implies that the parasite is capable of causing damage to the host. This type of a symbiotic association draws our attention because a parasite may become pathogenic if the damage to the host results in disease. Some parasitic bacteria live as normal flora of humans while waiting for an opportunity to cause disease. Other non indigenous parasites generally always cause disease if they associate with a non immune host.

History of Bacteriology

HISTORY OF BACTERIOLOGY

Bacteria existed long before mankind evolved and bacterial diseases are likely to have coevolved with each species. Many of the bacterial diseases that microbiologists encounter today have been around for as long as humans have e.g Leprosy in the Bible, and in some cases even longer, while others may have developed in recent years. However, doctors and scientists were unaware of the cause of infectious diseases for many centuries. With the beginning of the understanding of microbiology, the importance of bacterial pathogens became apparent.

Early speculations on disease
The idea that disease is caused by something too small to be seen occurs in various scripts before the modern age. In the first century BC the Roman author Varro speculated that some diseases might be caused by tiny animals carried in the air. Varro warned people to avoid marshy places during building work because he thought they contained invisible insects that produced disease by gaining access to the body via the mouth and nostrils.
 

The Greek physician Hippocrates (c 460–377 BC) put forward the notion that disease arose as a result of an imbalance of the four humours that were present in the human body. This imbalance was thought to be caused by miasmas – vapours from either rotting vegetables or rotting bodies, marshy places or polluted rivers (Miasmatic theory). Close contact with any of the above was believed to result in disease. Jacobo Forli dismissed the miasma theory of disease and claimed that it was not possible to get a disease by simply breathing in air alone. He stated that disease arose by breathing in contagious particles that floated in the air, and that these particles created the infection. However, belief in the miasma theory continued from before the first century, through the Roman and Greek periods, right up until around 1500
 

Early diagnostic techniques
Irrespective of whether early doctors subscribed to the miasma theory or the presence of contagious particles, one of the most common diagnostic techniques was uroscopy. The patient was required to produce a urine sample, which would be taken away and examined for appearance, smell and taste. Indeed, sweet tasting urine is a clue to the diagnosis of diabetes. Patients with jaundice will have brown urine and patients with kidney disease may have foamy or red urine. Bloody urine can denote tumours of the urinary tract while white urine can signify infection. Medical practitioners from the earliest times did not overlook these changes. Once the urine examination was complete, the observations were compared with the known colours, smells and tastes recorded on a so-called uroscopy wheel. The doctor would then arrive at a diagnosis and treat the patient accordingly.

From Fracastoro to the golden age
The examination of urine was practised until the time of Girrolamo Fracastoro, an Italian doctor and physiologist who published a paper in 1546 stating that diseases were caused by 'seminaria contagiosa', particles that could be transmitted by direct contact, touch or contaminated fomites. In this respect, Fracastoro’s work is the first evidence of someone thinking through germ theory and the fact that disease could be transmitted by contaminated objects. He also noticed that not all contagious particles were alike, as various diseases attacked different organs. He recorded that smallpox and measles occurred mostly in children, that everyone was attacked once but it was rare for people to have a second attack. Clearly, he recognised evidence of immunity. Fracastoro’s writings have been described as a great landmark in the history of infectious diseases.
 

Theory of spontaneous generation
Fracastoro's views, however, were not universally held. Most scientists and philosophers at that time thought that disease could occur as a result of spontaneous generation i.e some life could arise spontaneously from non living matter (abiogenesis). This theory was based on the observation that meat when left out in the open produced maggots, that mushrooms appeared on rotting wood, that rats and mice emerged from piles of litter and similar phenomena
 

Subsequently, the first serious attack on the idea of spontaneous generation was made in 1668 by Francesco Redi, an Italian physician and poet. At the time, it was widely held that maggots arose spontaneously in rotting meat. Redi, however, believed that maggots developed from eggs laid by flies. He tested his hypothesis by setting out meat in a variety of flasks, some of which were open to the air and some were sealed. As Redi expected, maggots appeared only in the open flasks where flies could reach the meat and lay their eggs.
The results of this experiment did not satisfactory Redi's critics, who claimed that fresh air was needed for spontaneous generation. Redi responded by setting up a second experiment, in which some jars were covered with gauze to allow air to circulate. No maggots appeared in the gauze-covered jars, even though air was present. Redi’s experiments notwithstanding, the belief in spontaneous generation remained strong. Interestingly, Louis Pasteur is charged with producing the first evidence that spontaneous generation does not occur and that bacterial infection comes from the environment, but clearly Francesco Redi was doing similar work in 1688, almost 200 years before Pasteur.
 

Power of the microscope
The debate over spontaneous generation continued for centuries. Around about the middle of 17th century microscopy became an important part of the history of bacteriology. Primitive microscopy was improved by three contemporaries: Robert Hooke in England, Antoni van Leeuwenhoek in The Netherlands, and Marcello Malphigi in Italy.
 

Hook's microscope was pretty advanced for its time, having a lens, eye tube and light source. van Leeuwenhoek’s microscope was almost as good, but it was he who received the praise because he was the first to describe bacteria morphologically and report them in a series of letters to The Royal Society in London. He reported seeing little living animals which he called ‘animalcules’ in water and from scrapings from his teeth. Microscopy revealed a whole new world of organisms that appeared to arise spontaneously.
More early experiments, more debate........
 

In 1745, English doctor John Needham suggested an experiment to test whether or not microorganisms appeared spontaneously after boiling. One of his findings was that if you take a broth (chicken or corn), boil it and then leave it in the open it will flourish with microorganisms. He therefore claimed that microbes developed spontaneously from the fluids. He published his work, believing that he had definite proof of spontaneous generation.
 

An Italian priest, Lazzaro Spallanzani, suggested 20 years later that perhaps the microorganisms had entered the broth from the air after the broth was boiled, but before it was sealed. To test his theory, he modified Needham's experiment. He placed the broth in a flask, sealed the flask, drew off the air to create a vacuum, and then boiled the broth. No microorganisms grew. Proponents of spontaneous generation however claimed that a ‘vital force’ necessary for spontaneous generation had been destroyed by the heat and was kept out of the flasks by the seals.
 

Golden era of microbiology
The French scientist Louis Pasteur finally disapproved the theory of spontaneous generation in 1861. Pasteur showed conclusively that spontaneous generation of life does not occur, and that microorganisms are everywhere, including in the air. To clarify that air and dust were the source of microbes, he filled flasks with broth and fashioned their openings into elongate, swan-neck shaped tubes. The flasks’ openings were freely open to the air but were curved so that gravity would cause any airborne dust particles to deposit in the lower part of the necks. He heated the flasks to sterilize the broth and then incubated them. As long as the flask remained intact, the broth remained sterile but if the neck was broken off so that dust fell directly down into the container, microbial growth immediately commenced. Pasteur was a pioneer of the ‘germ theory’ that disease was caused by living agents. After studying the causes of the souring of milk and wine, Pasteur concluded that these were due to fermentation and also observed that certain bacteria would only grow if oxygen was absent. He also demonstrated that heating could be used to kill microorganisms in milk and wine. This process is called pasteurization and is still in use today.
 

The English physicist John Tyndal while trying to confirm the results of Pasteur’s experiments determined the capacity of bacteria to exist in two forms: a heat labile form likely to be destroyed by heat and a heat resistant form that could survive high temperatures. He found out that intermittent heating at 1000C could eliminate viable microorganisms and thus sterilize solutions thereby validating Pasteur’s disproof of spontaneous generation. This intermittent heating on successive days is known as Tyndalization.
 

The British surgeon Joseph Lister was the first to introduce aseptic techniques after noticing that women became infected after getting examined by doctors coming directly from the autopsy room. He introduced disinfecting of hands and the air prior to surgery. In 1861, he noticed that patients with infected wounds whose dressings were contaminated with Penicillium mould showed greater improvement than those whose dressings were not contaminated. He concluded that the mould must have produced some form of antibacterial agent. Lister corresponded with Pasteur about this and used the term 'antibiosis'. In turn, Pasteur described to Lister how the peasants of Brittany and of the Russian steppes had, for hundreds of years, healed their sores with Penicillium mould. Thus, Pasteur and Lister demonstrated the possibility of using one microbe to kill another, an observation that predated Sir Alexander Fleming’s discovery by over half a century.
 

While the germ theory of disease was becoming established and the destruction of harmful bacteria investigated, it was inevitable that the new science of bacteriology would include a wave of research into isolating bacteria and differentiating them. The undisputed leader in this field was Robert Koch (1843–1910), a small-town medical officer in Poland. He wrote a letter to Ferdinand Cohn, Professor of Botany in Breslau, in April 1876 stating that he had observed the complete life cycle of the anthrax bacillus and that he was prepared to travel to Breslau to demonstrate this to Cohn and his colleagues. Using blood serum from cattle and the aqueous humour from excised animal eyeballs (immediately after slaughter) as a culture fluid, and placed the inoculated preparation under sealed coverslips on a warm stage and studied the progress of the culture hour by hour. He demonstrated the relevance of the organism to the disease by animal inoculation and he showed that the disease was transmissible from one animal to another for over 20 generations. Koch published this work under Cohn's guidance in late 1876.
 

The next person to make an impact on the development of microbiology as a science was Richard Petri, who, in 1887, invented the dish that bears his name. Petri described it as "a slight modification of the culture method”! There was no gold standard culture medium at the time, and people were trying various things. Koch, for example, soaked raw potato in "corrosive sublimate" then sterilised it in steam, split it in two with a sterile knife and allowed the halves to fall apart in a previously sterilised glass dish. The surfaces of the potato were then inoculated. The solidification of broth using gelatin was suggested by mycologist Oscar Bredfeld, who was visiting Koch at that time. Bredfeld suggested using a liquid mixture of bacteria diluted with sterile medium and obtaining growth from a single bacterial cell. Subsequent solidification with gelatin produced early poured plates and gave rise to agar plates.
 

Robert Koch also discovered and cultured the bacteria that caused several diseases, including tuberculosis, during the latter part of the 19th century. He used agar as a solidifying agent, allegedly on the advice of Frau Hesse, the wife of a colleague, to prepare a solid medium on which bacteria could be grown in individual colonies and therefore in pure culture. Furthermore, he put forward his famous criteria – Koch’s postulates – for confirming an organism as the cause of a specific disease. These postulates were elegantly confirmed by his assistant, Loeffler, a few years later when working with diphtheria. Thus, Koch gained a reputation for accurate, detailed and painstaking work.
 

With the introduction of solid media, Koch was able to recover the TB bacillus from sputum. The discovery that he could inoculate animals with the isolated bacteria and cause similar disease followed by reisolation of the infection from the animal revealed an important principle of pathogenicity.