Section Outline
- 1. Introduction and Bacterial Taxonomy
- 2. Morphology and Physiology of Bacteria
- 3. Sterilization and Disinfection
- 4. Culture Media and Culture Methods
- 5. Identification of Bacteria
- 6. Bacterial Genetics
- 7. Antimicrobial Agents, Antimicrobial Resistance and Antimicrobial Susceptibility Testing
Chapter Preview
- • History
- • Bacterial taxonomy
Medical microbiology is a branch of medicine that deals with the study of microorganisms and their role in human health and diseases. It also concerns with the diagnosis, treatment and prevention of various infectious diseases. There are four kinds of microorganisms that cause infectious disease: bacteria, fungi, parasites and viruses. The branches of medical microbiology are as follows:
- General microbiology: Study of general properties of microorganisms, such as bacterial morphology, sterilization and disinfection, culture identification methods, bacterial genetics, etc.
- Immunology: The study of the immune system
- Bacteriology: The study of bacteria
- Virology: The study of viruses
- Mycology: The study of fungi
- Parasitology: The study of parasites; it has two arms
- Protozoology: The study of protozoa
- Helminthology: The study of helminths.
HISTORY
The existence of microorganisms was hypothesized for many centuries before their actual discovery. The teaching of Mahavira (Jainism, 6th century BC) and the postulation of Varo and Columella (who named the invisible organisms as ‘Animalia minuta’) were some of those attempts.
CONTRIBUTORS IN MICROBIOLOGY
Antonie Philips van Leeuwenhoek (1676)
He was the first scientist who observed bacteria and other microorganisms, using a single-lens microscope constructed by him and he named those small organisms as ‘Little animalcules’ (Fig. 1.1A).
Edward Jenner
Edward Jenner 1796, developed the first vaccine of the world, the smallpox vaccine. He used the cowpox virus (Variolae vaccinae) to immunize children against smallpox from which the term ‘vaccine’ has been derived. The same principles are still used today for developing vaccines.
Louis Pasteur
Microbiology developed as a scientific discipline from the era of Louis Pasteur (1822–1895). He is also known as father of microbiology. He was a professor of chemistry in France. His studies on fermentation led him to take interest to work in microbiology (Fig. 1.1B). His contributions to microbiology are as follows:
- He had proposed the principles of fermentation for preservation of food.
- He introduced the sterilization techniques and developed steam sterilizer, hot air oven and autoclave.
- He described the method of pasteurization of milk.
- He had also contributed for the vaccine development against several diseases, such as anthrax, fowl cholera and rabies.
- He disproved the theory of spontaneous generation of disease and postulated the ‘germ theory of disease’. He stated that disease cannot be caused by bad air or vapor, but it is produced by the microorganisms present in air.
- Liquid media concept: He used nutrient broth to grow microorganisms.
- He was the founder of the Pasteur Institute, Paris.
Joseph Lister
Joseph Lister (1867) is considered to be the father of antiseptic surgery. He had observed that postoperative infections were greatly reduced by using disinfectants such as diluted carbolic acid during surgery to sterilize the instruments and to clean the wounds.4
Robert Koch
Robert Koch provided remarkable contributions to the field of microbiology. He was a German general practitioner (1843–1910) (Fig. 1.1C). His contributions are as follows:
- He introduced solid media for culture of bacteria, Eilshemius Hesse, the wife of, one of Koch's assistants had suggested the use of agar as solidifying agents.
- He also introduced methods for isolation of bacteria in pure culture.
- He described hanging drop method for testing motility.
- He discovered bacteria such as the anthrax bacilli, tubercle bacilli and cholera bacilli.
- He introduced staining techniques by using aniline dye.
- Koch's phenomenon: Robert Koch observed that guinea pigs already infected with tubercle bacillus developed a hypersensitivity reaction when injected with tubercle bacilli or its protein. Since then, this observation was called as Koch's phenomenon.
- Koch's postulates: Robert Koch had postulated that a microorganism can be accepted as the causative agent of an infectious disease only if four criteria are fulfilled. These criteria are as follows:
- The microorganism should be constantly associated with the lesions of the disease.
- It should be possible to isolate the organism in pure culture from the lesions of the disease.
- The same disease must result when the isolated microorganism is inoculated into a suitable laboratory animal.
- It should be possible to re-isolate the organism in pure culture from the lesions produced in the experimental animals.
An additional fifth criterion was introduced subsequently which states that antibody to the causative organism should be demonstrable in the patient's serum.
Exceptions to Koch's postulates: It is observed that it is not always possible to apply these postulates to study all the human diseases. There are some bacteria that do not satisfy all the four criteria of Koch's postulates. Those organisms are:
- Mycobacterium leprae and Treponema pallidum: They cannot be grown in vitro; however, they can be maintained in experimental animals.
- Neisseria gonorrhoeae: There is no animal model; however, it can be grown in vitro.
Paul Ehrlich
Paul Ehrlich (1854–1915) was a German scientist and is also known as father of chemotherapy (Fig. 1.1D). His contributions are as follows:
- He was the first to report the acid-fast nature of tubercle bacillus.
- He developed techniques to stain tissues and blood cells.
- He proposed a toxin-antitoxin interaction called Ehrlich phenomenon and also introduced methods of standardising toxin and antitoxin.
- Chemotherapy: He discovered salvarsan, an arsenical compound (also called as the ‘magic bullet’) as the first effective medicinal treatment for syphilis, thereby initiating and also naming the concept of chemotherapy.
- The bacteria ‘Ehrlichia’ was named after him.
- In 1908, he received the Nobel prize in Physiology or Medicine for his contributions to immunology.
- He was the founder and first director of what is known now as the Paul Ehrlich Institute, Germany.
Other Important Contributors
- Hans Christian Gram (in 1884): He developed a method of staining bacteria which was named as ‘Gram stain’ to make them more visible and differentiable under a microscope.
- Charles Chamberland: He is one of Pasteur's associates, constructed a porcelain bacterial filter in 1884 by which the discovery of viruses and their role in disease was made possible. The first viral pathogen to be studied was the tobacco mosaic virus.
- Ernst Ruska: He was the founder of electron microscope (1931).
- Alexander Fleming (in 1929): He discovered the most commonly used antibiotic substance of the last century, i.e. penicillin.
- Goodpasture: He described the viral culture technique in chick embryo.
- Lady Mary Wortley Montagu: Introduced variolation for immunizing against smallpox.
- Elie Metchnikoff: He described phagocytosis and termed phagocytes.
- Emmy Klieneberger (1941): She described the existence of L forms of bacteria.
- Barbara McClintock: She described the mobile genetic elements in bacteria called transposons.
- Walter Gilbert and Frederick Sanger were the first to develop (1977) the method of DNA sequencing.
- Karry B Mullis: Discovered polymerase chain reaction (PCR) and was awarded Noble prize in 1993.
Discovery of Bacterial Agents
Several bacteria were discovered by many scientists (Table 1.1). The names of some of the bacteria are coined in the honor of scientists who discovered them (Table 1.2).
Nobel Laureates
A number of scientists in medicine or physiology have been awarded Nobel prizes for their contributions in microbiology (Table 1.3).
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BACTERIAL TAXONOMY
Bacterial taxonomy comprises of three separate but interrelated important areas.
- Classification: It refers to hierarchy based arrangement of bacteria into taxonomic groups or taxa (singular, taxon) on the basis of similarities or differences in their biochemical, physiological, genetic, and morphological properties.
- Nomenclature: It refers to the naming of taxa according to their characteristics, by following the international rules.
- Identification: It refers to the practical use of a classification scheme such as: (1) Identification of an unknown taxon by comparing with a defined and named taxon, (2) To isolate and identify the causative agent of a disease.
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BACTERIAL CLASSIFICATION
The most recent taxonomic classification of bacteria is based on Cavalier and Smith's six kingdoms classification (1998). It is the most accepted classification at present, surpassed the previous five kingdom classification (Whittaker, 1969) and three domain classification (Woese, 1990) (Table 1.4).
Cavalier and Smith's Classification
It is a molecular classification, which divides all living structures of the earth into six kingdoms—Bacteria, Protozoa, Chromista, Plantae, Fungi and Animalia.
Kingdom Bacteria is divided successively in decreasing order of hierarchy into phylum/division, class, order, suborder, family, tribe, genus and species. For example, the full taxonomical position of Escherichia coli is given in Table 1.5.
Principle Used to Classify Bacteria
There is no universally accepted principle to classify bacteria. There are mainly three approaches, phylogenetic, Adansonian and molecular.
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Phylogenetic Classification
This is a hierarchical classification representing a branching tree-like arrangement; one characteristic (or trait) is being employed for division at each node of the tree (Fig. 1.2).
- This system is called phylogenetic because it implies an evolutionary arrangement of species.
- Here, the characteristics are arbitrarily given special weightage. Depending on the characteristic so chosen, the classification would give different patterns.
- The characteristics which are given importance depend up on various properties of the organisms such as:
- Morphology of bacteria—cocci or bacilli.
- Staining property such as gram-positive and gram-negative.
- Biochemical reactions, e.g. coagulase positive Staphylococcus and coagulase negative Staphylococcus.
- Antigenic structure, e.g. antigenic structure of somatic antigen present in bacterial cell wall.
- Though, this classification is a convenient and user friendly method, but it is not a perfect method. Because the weighted characters used may not be valid all the time for a given bacterium. For example, fermentation of lactose though is an important property to classify family Enterobacteriaceae; but is not a permanent trait. In due course of the time, bacteria may lose or gain the property to ferment lactose.
Adansonian Classification
To avoid the use of weighted characteristics, Michel Adanson proposed another method (1774) that classifies organisms based on giving equal weight to every character of the organism.
- This is also called phenetic classification. It has its greatest application in numeric taxonomy.
- Numerical taxonomy: The concept was first developed by Robert R Sokal and Peter HA Sneath in 1963.
- With the advent of computer facilities, the principle of phenetic classification has been extended further so that very large numbers of characters of several organisms can be compared at the same time.
- They have created a taxonomic system by using numeric algorithms like cluster analysis rather than using subjective evaluation of their properties which are arbitrarily given special weightage.
Molecular Classification
It is based on the degree of genetic relatedness of different organisms. Guanine + cytosine (G + C) content of bacteria is estimated after extracting DNA from pure bacterial culture. The nucleotide base composition and the base ratio vary widely among different groups of microorganisms, but for any one particular species, it is constant.
NOMENCLATURE
Nomenclature is the branch of taxonomy, that is concerned with designating scientific names to taxa, based on a particular classification scheme and in accordance with agreed international rules and conventions.
- Bacterial nomenclature also follows the same rules as proposed by Swedish botanist Carolus Linnaeus who invented the modern system of binomial nomenclature.
- Scientific names for taxonomic levels above genus are always capitalized but not italicized; for example, Phylum Proteobacteria.
- In binomial nomenclature system, the scientific name of bacteria comprises of a genus name (starts with a capital letter) and species name. Both genus and species should be written in italic or are underlined; e.g. Staphylococcus aureus or Staphylococcus aureus.
- The genus (plural: genera) is usually a Latin noun whereas the species refers to a defined taxon of organisms within a particular genus.
- The genus and species are coined based on some property of the bacteria; for example,
- Staphylococcus aureus is named after their arrangement in cluster (Staphyle means as bunch of grapes) and type of pigmentation they produce (aureus meaning golden yellow)
- Neisseria meningitidis is named after—the discoverer (U. Neisser) and the disease it causes (meningitis).
- Brucella suis and Brucella melitensis (named after the discoverer (Brucella from David Bruce) and the animal host (suis meaning pig) and the place of discovery (melitensis from Malta, Europe).
- Typing: The species can also be classified further by various typing methods as described in Chapter 5.
Type Cultures
There are many international reference laboratories which are designated as type culture reference centers.
- They maintain the representative cultures of the established species, which show all the standard characteristics of the original strain.
- The strains isolated in the laboratories are compared using the standard strains supplied by these type culture centers.
- The original cultures of any new species described are deposited in type collection centers.
- The two most important type collection centers of the world are:
- ATCC (American Type Culture Collection), USA
- NCTC (National Collection of Type Cultures), UK.
I. Write short notes on:
- Bacterial taxonomy
- Contributions of Louis Pasteur in the field of microbiology
- Contributions of Robert Koch in the field of microbiology