Category:Bacteria - Overview
Introduction
A typical bacterial cell is composed of an outer capsule, a cell wall, a cell membrane, cytoplasm containing nuclear material and ifmotile, appendages such as flagella and fimbrae or pili. Some species of bacteria are more resistant to environmental influences than others, particularly those species of bacteria that are able to produce spores which can remain inactive until the appropriate environmental conditions prevail allowing the bacteria to resist conditions such as freezing, wet, dry or hot
conditions.
The structural features of pathogenic bacteria are important in the production of disease and also very useful for the identification and diagnosis of infection in veterinary medicine.
Structure of Bacterial Cell
Capsule
The outermost part of a bacterial cell is the capsule, often described as the glycocalyx. Most capsules are composed of polysaccharides, although in some species the capsule is made of polypeptides. Capsules can be visualised
under light microscopy by using staining techniques.
The main function of bacterial capsules is to provide protection from adverse environmental conditions, prolonging the period of survival in such conditions. The capsule also facilitates adherence to surfaces and interferes with host cells involved in phagocytosis.
Cell Wall
The cell wall lies between the cell membrane (inner) and the capsule (outer) and protects the bacteria from mechanical damage and osmotic lysis. Cell walls are non-selectively permeable and are only able to exclude large molecules. Species dependant differences in the structural and chemical composition of the cell wall creates variation in the pathogenicity of the cell and also influences the staining properties of the cell which is important for species identification. Peptidoglycan (a polymer unique to prokaryotic cells) provides the cell wall with rigidity.
Bacteria can be divided into two major groups on the basis of the colour of the cell wall when stained using the Gram Method. The groups are called “Gram Positive” and “Gram Negative”. Gram positive bacteria stain blue and have a thick cell wall composed mainly of peptidoglycan and teichoic acids. Gram negative bacteria stain red and their walls have a much more complex structure containing
an outer membrane, a periplasmic space and an inner membrane. For further information on the structure of both types of cell wall please see Bacterial structure
Antibiotic treatments such as penicillin interefere with the ability of the bacterial cell to produce peptidoglycan and therefore cannot produce their cell wall making them more vulnerable to the environment.
Cytoplasmic Membrane
Bacterial cytoplasmic membranes are flexible structures composed of phospholipids and proteins and are similar to the lipid bi-layer membranes found in eukaryotic cells. Only a limited number of small molecules such as water, carbon dioxide and lipid-soluable compounds can enter bacterial cells by passive diffusion. Nutrients and waste metabolites are transferred via active transport using ATP (adenosine triphosphate).
The cytoplasmic membrane is also the site of electron transport for bacterial respiration and also contains enzymes and carrier
molecules that function in the biosynthesis of DNA, cell wall polymers and membrane lipids.
Cytoplasm
The cytoplasm is enclosed by the cytoplasmic membrane and is an aqueous fluid containing nuclear material, ribosomes, nutrients and the enzymes involved in most cellular functions. Storage granules can often be seen in the cytoplasm under certain environmental conditions. Storage granules mainly contain starch and glycogen.
Nuclear Material
The bacterial genome is composed if a single haploid circular chromosome containing double-stranded DNA (dsDNA). Bacterial genomes
vary in size depending on species but often has a folded structure to form a dense body which is visible using a scanning electron microscope. During replication the DNA helix unwinds and both daughter cells (produced by binary fission) receive
a copy of the original genome.
The cytoplasm also contains Plasmids. Plasmids are small circular pieces of DNA that are separate from the genome and are capable of autonomous replication. Several different plasmids can be within the cytoplasm of a single bacteria. Plasmids
can be transferred between bacteria during binary fission or through a process called conjugation. Plasmid DNA codes for characteristics including antibiotic resistance and endotoxin production.
Flagella & Pili/Fimbrae
Motile bacteria have flagella allowing them to move into suitable micro-environments in response to physical or chemical stimuli. It is mainly gram negative bacteria that possess flagella and they are rarely present in cocci species. Flagella are normally several times longer than the bacterial cell and are composed of the protein flagellin. Flagella are usually anchored
to the cell wall.
Pili are straight, hair-like appendages composed of pilin and also anchored to the cell wall. Pili are most common on gram negative
bacteria. In pathogenic bacteria pili function as adhesions for receptors on mammalian cells.
Endospores
Endospores are dormant bodies that are highly resistant to the environment. The only genera of pathogenic bacteria that are able to
produce endospores are Bacillus and Clostridium. Endospores have a “spore coat” and are effectively in a dehydrated state with negligible metabolic activity. Due to the thermostability of endospores they can only be destroyed with certainty by moist heat at 121C for 15 mins.
An endospore will reactivate in response to environmental factors such as exposure to heat, abrasion of the spore coat or environmental acidity. Reactivation occurs in three stages; activation, initiation and outgrowth. In the correct conditions, germination will occur in which the spore coat is degraded and water is absorbed.
Bacterial Growth and Measurement
Bacterial Growth
Appropriate environmental conditions are needed for bacterial growth including moisture, pH, temperature, osmotic pressure,
atmosphere and nutrients. The time required for the production of daughter cells (generation time) is dependant on genetic and nutritional factors. Generation time can range from 20 mins to 20 hours.
Bacterial Nutrition
Bacterial growth requires nutrients to be available in the immediate environment. Bacteria are mainly chemoheterotrophs (using organic chemicals as energy). Specialised forms of agar plate containing different types of media have been developed to facilitate bacterial growth in controlled specific conditions. Therefore it is possible to determine the species of bacteria based on, amongst other things, the type of media they are able to grow on.
Most pathogenic bacteria can be grown on a nutrient medium at 37C, although they can grow between 20-45C. Most bacteria also grow optimally at a neutral pH and it is standard practice to buffer culture media so maintain it around pH 7. Another key determinant in bacterial growth is their preference for different types of atmosphere. Bacterial preference for oxygen means it is possible to assign all bacteria into four main groups; aerobes, anaerobes, facultative anaerobes and microaerophiles. Therefore anaerobic bacteria are unable to grow in an atmosphere containing oxygen.
Methods for Counting bacteria
| Method | Technique | Comments |
|---|---|---|
| Microscopic Counting | ||
| Direct Smear | Smear on microscope slide from defined volume and dilution. Count performed using 50 microscope fields. | Slow and unreliable method and cannot differentiate viable and non-viable bacteria |
| Chamber Counting | Count fixed volume of bacterial suspension using a calibrated slide | No differentiation between viable and non-viable bacteria |
| Colony Counting | ||
| Spread Plate | Known vol of bacterial suspension spread onto agar plate and incubated for 24-48 hours | Number of colonies counted and expressed as colony-forming units (CFU)/ml of suspension |
| Pour Plate | A small vol of a known bactrial dilution is added to a petri dish with 20ml of molton agar at 45-48C and mixed | Colony counting carried out as above. |
| Miles-Misra | Diluted bacterial solution placed on plate in 5 seperate positions | Number of colonies counted and expressed as colony-forming units (CFU)/ml of suspension |
| Membrane Filtration | Following filtration of a known vol of bacterial dilution through a 0.22um pore size, filter is placed on an agar plate and incubated for 24-48 hours | The number of viable bacteria are expressed as CFU/ml of fluid |
| Other Counting Methods | ||
| Opacity Tubes | A bacterial suspension is matched visually with Mcfarland's opacity standard tubes | This test indicates the total bacterial cell numbers per ml |
| Electronic Counting | Counting machines such as the Coulter Counter can give rapid and accurate results | Reliability of results is dependant on quality control and test only gives a total cell count. |
Bacterial Genetics
Replication of Bacteria
Bacteria are haploid and have one circular chromosome of double stranded DNA. Bacteria replicate through binary fission producing genetically identical daughter cells. Each molecule of DNA in the daughter cells is composed of a strand from the parent and a newly synthesised complementary strand. This process of DNA replication is called semiconservative replication.
Plasmids
Plasmids are small pieces of genetic material found in the cytoplasm and these plasmids are able to replicate independantly of the bacterial chromosome. Most species of bacteria contain plasmids that are composed of double stranded DNA which are circular in shape. In pathogenic bacteria it is often the plasmid that encodes virulence factors and traits such as antibiotic resistance.
Replication of most plasmids is not directly related to the replication of the host bacterium and it has been found that the distribution of plasmids to daughter cells is a random process as plasmids in the cytoplasm may or may not be transferred when the cytoplasm of the cell is seperated prior to forming the daughter cells.
Bacterial plasmids can not only be transferred during bacterial replication but also via processes called conjugation and transformation (although the transformation process rarely occurs in nature).
Bacteriophages
A bacteriophage is a term used to describe a virus that is able to infect a bacterial cell and they can be either virulent or temperate depending on their method of replication. Virulent bacteriophages undergo a lytic cycle within the bacterium which eventually results in the production of bacteriophage progeny from the cell and the lysis of the bacterium.
A bacteriophage can be composed of either DNA or RNA and iehter single or double stranded. The capsid (outer protective layer) of the bacteriophage often remains outside the bacterial cell after the viral nucleic material has entered the cell cytoplasm. The host specificity of bacteriophages is related to the chemical affinity between attachment structures on the surface of the bacteriophage capsid and the receptors on the surface of the bacterium.
Genetic Variation
Genetic variation can occur in a number of ways and the genotype of the bacteria determines its inheritable potential. Below are the main ways that genetic mutation can occur in bacteria;
Mutation
A mutation is a stable inheritable alteration in the bacterial genome. This means that base pairs within a gene are altered. Genes with altered base pairs may, or may not, depending on the mutation be functional or can incorrectly code for an amino acid in a protein resulting in a phenotypic change rather than simply a gene alteration. The type of mutations occurring in bacteria are silent, non-sense, mis-sense, frame shift, deletion of base pairs, insertions, translocations and inversions.
Genetic Recombination
Genetic recombination occurs when sequences of DNA from seperate sources become integrated. This new genetic material can be introduced via conjugation, transduction and transformation.
Conjugation
Conjugation represents the process whereby DNA is transferred from a donor cell to a recipient cell. The donor cell synthesises a modified "pilus" which the donor cell inserts into the recipient cell. This is often called a sex pilus. Genetic material is then transferred through the pilus to the recipient. During conjugation, plasmid genetic material is mostly transferred, although chromosomal DNA can also be transferred via this process. Conjugation is most frequently associated with gram negative bacteria, but can occur in some gram positive bacteria. A sex pilus is not formed in gram positive bacteria and instead plasmid DNA is transferred when the bacteria are in close physical contact.
Tranduction
During transduction, DNA from a donor bacterium is incorporated into the nucleic acid of a bacteriophage and it is the progeny of the bacteriophage infecting another bacterium that allows the transfer of the genetic material.
Transformation
The process of transformation involves the transfer of genes from a segment of chromosomal DNA from a lysed donor bacterium to a fully functional recipient. Natural transformation is uncommon and is usually restricted to propcedures carried out in the lab.
Laboratory Diagnosis of Bacterial Disease
Laboratory techniques are often required for identifying the aetiological agent and/or the antimicrobial susceptibility of pathogens. It should be noted that any laboratory analysis should be accompanied by a full clinical examination and history.
Identification of Pathogenic Bacteria
Pathogenic bacteria can be identified by the examination of stained smears, cultural and biochemical characteristics and detection by immunological and molecular methods.
Stained Smears
There are a number of different routine methods used to stain bacteria for examination on a microscope slide. Gram stain smears are rapid and able to detect bacteria in large numbers and is often used for a 'rough and ready' analysis of tissue samples. Below is a table showing most of the main stains;
| Staining Method | Comments |
|---|---|
| Gram Stain | Most common in bacterial smears. The stain contains crystal violet which is retained in the cell wall of the bacterium. Gram positive bacteria are blue and gram negative bacteria do not retain the crystal violet and appear red, the colour of the counterstain. |
| Giemsa | Used to identify Dermatophilus congolensis, rickettsiae and Borrelia species which stain blue. |
| Dilute carbol fuchsin | Used for identifying Campylobacter species, Brachyspira species and Fusobacterium species which stain red. |
| Polychrome methylene blue | Used to identify Bacillus anthracis in blood smears which stain blue with distinctive pink capsules |
| Ziehl-Neelsen stain | Red staining bacteria are described as acid-fast or Ziehl-Neelsen positive |
Bacterial Culture
As noted above, the selection of the culture medium, atmospheric conditions and pH are among many variables that need to be considered for the successful culture of bacteria in the lab. A routine culture undertaken involves using a combination of blood agar (see below) and MacConkey agar (see below) together with incubation for between 24-48hours. Blood agar is able top support most pathogenic species of bacteria and is usually appropriate for routine primary isolation. Selective media is then normally used for particular organisms.
Agar plates should be inoculated using a streaking technique facilitating growth of isolated colonies. The aseptic technique of inoculation should also be used to prevent contamination.
Below is a table detailing the main types of medium used in bacterial culture;
| Medium | Comments |
|---|---|
| Nutrient Agar | Most commonly used basic medium. Non-fastidious bacteria (unable to produce their own vitamins) can grow on this medium. This medium is also suitable for demonstrating colonial morphology and pigment production. This type of agar is also commonly used as part of bacterial counting techniques as described above. |
| Blood Agar | This medium contains blood and is able to support the growth of pathogenic bacteria. This medium also allows the recognition of bacterial haemolysin production |
| MacConkey Agar | A selective medium containing bile which is useful for the isolation of enterobacteria and other gram negative bacteria. This medium also allows differentiation of lactose and non-lactose fermenting species. Colonies of lactose fermenters turn the surrounding medium pink as the medium also has a pH indicator. |
| Selenite broth, Rappaport-Vassiliadis Broth | Selective enriched medium used to isolate salmonellae from samples containing other gram negative enteric organisms |
| Edwards Medium | A blood agar based selective medium used for the isolation and recognition of steptococci |
| Chocolate Agar | Heat-treated chocolate agar which provides special growth factors for the isolation of Haemophilus species and for the culture of Taylorella equigenitalis. |
| Brilliant green agar | Indicator medium for the presumptive identification of Salmonella species. Salmonella colonies and surrounding medium have a pink appearence. |
| Buffered peptone water | Non-selective medium used for isolation of pathogens when present in low numbers in samples collected from foods and environmental sources |
Biochemical techniques
Biochemical tests relate to the catabolic activities of bacteria and use this to demonstrate the utilisation of particular substrates. The range of sugars utilised by bacteria is relatively small and therefore catabolism of sugars is often used as a method of identification. Commercial testing kits are available which usually consist of a strip of plastic cupules containing a test to which a suspension of the bacterium is added. The identity of the bacteria is then deduced from the pattern of the various cupules.
Below is a table of the commonly used biochemical tests;
| Test | Pathogens | Comments |
|---|---|---|
| CAMP Reaction | Steptococcus agalactiae, Rhodococcus equi, Actinobacillus pleuropneumoniae, Listeria monocytogenes | Haemolysis caused by Staphylococcus aureus is enhanced by pathogenic bacteria growing close to staphylococcal colonies |
| Pitting of Loeffler's serum slope | Arcanobacterium pyogenes | Proteolytic digestion of the medium around colonies |
| Haem-agglutination | Bordetella bronchiseptica | Agglutination of suspended ovine red blood cells by the bacteria |
| Nagler Test | Clostridium perfringens | Breakdown of lecithin in egg yolk agar by alpha toxin (lecithinase) produced by the organism. Surface application of antitoxin inhibits the alpha toxin activity |
Immunological Techniques
Immunological identification or serotyping uses the surface antigens on bacteria. Fluorescent antibody staining, antigen capture and direct enzyme-linked immunosorbent assays (ELISA) have been developed to identify bacterial pathogens. In all of these techniques the bacteria is bound by a specific antibody which has some form of indicator attached such as colour change enzymes or fluorescence.
Bacteriophage Typing
Some bacteriophages are very species specific and therefore phage typing represents another method that can be used to identify species of bacteria. This method allows bacterial species to be sub-divided into subtypes which are defined by their susceptibility to particular phages. Phage typing is commonly used to differentiate between Staphylococcus aureus and Salmonella enterica sub species.
Molecular Techniques
The most common molecular technique used to identify species of pathogenic bacteria are nucleic acid hybridisation and polymerase chain reactions (PCR). Nucleic acid hybridisation uses synthetic nucleic acid probes (specific for a particular species) that are applied to genetic material extracted from the pathogen. Probes can be used to detect DNA and RNA.
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