Bacteriophages

 Bacteriophages

Bacteriophages, or phages are viruses that infect bacteria. Bacteriophages were discovered independently by Frederick W. Twort and Félix d’Hérelle.  D’Hérelle coined the term bacteriophage, meaning “bacteria eater,” to describe the agent’s bacteriocidal ability.  Phage means ‘to eat’.  Phages occur in nature in close association with bacteria and can be isolated from feces, sewage and other natural sources of mixed bacterial growth.  Thousands of varieties of phages exist, each of which may infect only one type or a few types of bacteria or archaea.

Morphology

Phages consist of nucleic acid surrounded by a protein capsid.

The head consists of a tightly packed core of nucleic acid (DNA or RNA) surrounded by a protein coat or capsid. The size of the head varies in different phages from 28 nm to 100 nm. The nucleic acid may be either DNA or RNA and may be double-stranded or single-stranded. The capsid is made up of subunits known as Capsomeres.  The capsomeres consists of a number of protein subunits called Protomers. 

The tail is composed of a central hollow core or tube, a contractile sheath surrounding the core and a terminal base plate to which tail fibers or prongs (tail pins) or both are attached.  These tail fibers or prongs help the phage to bind to specific receptor sites on the bac­terial surface.

Type of nucleic acid present in the phage varies, some phages have DNA while some other phages carry RNA as genome. 

Bacteriophage T4                          Prescott

There are three basic structural forms of phage: an icosahedral head with a tail, an icosahedral head without a tail and a filamentous form.

       I.            Icosahedral or cubical symmetry – examples are φX174, MS2

    II.            Icosahedral head with a tail or Binal symmetry– examples are T2, T4, T6

 III.            Filamentous or helical symmetry –Examples are M13, fd

Bacteriophages are grouped into seven morphological types.

Type	Shape	Nucleic Acid	Example
A	Hexagonal head, rigid tail with a contractile sheath and tail fibres	Double stranded DNA	T2, T4, T6
B	Hexagonal head, flexible tail, no contractile sheath and may or may not have tail fibres	Double stranded DNA	T1, T5
C	Hexagonal head, shorter tail, no contractile sheath and may or may not have tail fibres	Double stranded DNA	T3, T7
D	Head made up of large capsomeres, no tail	Single stranded DNA	S13, φX174
E	Head made up of small capsomeres, no tail	Single stranded RNA	f2, MS2
F	Filamentous	Single stranded DNA	fd, f1
G	Pleomorphic, No capsid	Double stranded DNA	MV-L2

 

Life cycle of bacteriophages

During infection a phage get attached to a bacterium and inserts its genetic material into the bacterial cell.   After this, the phage usually follows one of two life cycles, lytic (virulent) or lysogenic (temperate).

Lytic phages take over the machinery of the cell to make phage components. They then destroy, or lyse, the cell, releasing new phage particles.

Lysogenic phages incorporate their nucleic acid into the host cell chromosome and replicate with in it as a unit without destroying the cell. Under certain situations, lysogenic phages get induced to follow a lytic cycle.

Lytic Cycle

During Lytic Cycle, the virulent phage replicate through the following stages - adsorption, penetration, transcription, assembly, maturation and release of progeny phage particles.

Example of a virulent phages are T2, T4, T6 phages of E coli

i. Adsorption

Phage particles attach to virus-specific receptors on the host cell by its tail. Adsorption is a specific process and depends on the presence of complementary chemical groups on the receptor sites on the bacterial surface and on the terminal base plate of the phage.

Initial adsorption of phage to the receptor is reversible, since only tips of tail fibers are attached to the bacterial cell surface.  Then the tail pins attach and the adsorption becomes irreversible. 

Adsorption is a very rapid process and it will be complete within minutes under optimal conditions. Any component on the bacterial surface can serve as receptor for some phage. Bacterial receptor may be part of the LPS, flagella, pili, membrane or wall carbohydrates or proteins.  Host specificity of phages is determined at the level of adsorption.

ii. Penetration

After adsorption, most phages inject their nucleic acid into the bacterial cytoplasm and leave their protein cap­sid outside, similar to injec­tion through a syringe.

During penetration, the tail fibers attach firmly to the cell and firmly attach the phage plate to cell wall.  The contractile tail sheath contracts and this will force the hollow interior tail tube into the bacterial cell wall. The phage DNA then passes through the tail tube. The empty phage head and tail remain outside the bacterium. Penetration may be facilitated by the presence of lysozyme on the phage tail that causes localized digestion of cell wall surfaces.    

Some phages such as T1 and T5 do not have contractile sheath and they inject nucleic acid through adhesion sites between inner and outer membranes of bacterial cell wall.  Filamentous and rod shaped bacteriophages enter bacterial cells and then release DNA in to cell.

iii. Transcription - Synthesis of Phage Nucleic Acid and Proteins

The synthesis of the phage components occurs immediately after penetration of the phage nucleic acid.  It occurs through several stages – formation of immediate early, delayed early and late gene products. 

Immediate early phage genes are transcribed by bacterial RNA polymerases.  Examples for the products are Nucleases to break down host DNA and enzymes to alter bacterial RNA Polymerase. 

Delayed early phage genes products include enzymes to produce Phage constituents, Phage polymerase, ligase and RNA polymerase.

Late gene products include structural components for new phage particles – heads, tails, fibers, etc. and phage lysozyme to lyse bacterial cell for releasing mature phage particles.

iv. Assembly and Maturation

After the synthesis of structural proteins and nucleic acids, the phage components begin to assemble into mature phages.  Phage DNA is condensed into a compact polyhedron and packaged into the head and then the tail struc­tures are added. This assembly of the phage compo­nents into the mature phage particle is known as maturation.

v. Release

Release of the mature progeny phages typically occurs by lysis of the bacterial cell. Phage enzymes act on the bacteri­al cell wall causing it to burst or lyse resulting in the release of mature daughter phages.

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Eclipse Phase, Latent Period and burst size

The interval between the entry of the phage nucleic acid into the bacterial cell and the appearance of the first infectious intracellular phage particle is known as the eclipse phase. It represents the time required for the synthesis of the phage components and their assembly into mature phage particles.

The interval between the infection of a bacterial cell and the first release of infectious phage particles is known as the latent period.

The average yield of progeny phages per infected bacterial cell is known as the burst size (100 to 300 phages).

Lysogenic Cycle

The Lytic or Virulent phages produce lysis of the host cell, while the Lysogenic or Temperate phages enter into a symbiotic relationship with their host cell without destroying the host cell. Lambda phage (λ phage) that infect E coli is an example.

When a temperate phage attacks a bacterium, two things may happen.  In some infected cells, the phage multiplies and a lytic cycle occurs.  In most of the other infected cells, the multiplication of the phage does not happen, it is repressed by a repressor protein.  In this situation, after entry into the host cell, the temperate phage nucleic acid gets integrated into the bacterial chromo­some. The integrated phage nucleic acid is known as the prophage.  Here the prophage becomes and behaves like an integral part of the host chromosome.  As the bacterium reproduces, viral nucleic acid also gets replicated along with bacterial chromosome and is transmitted to the daughter cells.  This phenomenon is called lysogeny and a bacteri­um that carries a prophage within its genome is called a lysogenic bacterium or lysogen.

Under certain natural conditions or under artificial stimuli such as exposure to certain physical and chemical agents such as UV rays, hydrogen peroxide and nitrogen mustard, the prophage may become ‘excised’ from the bacterial chromosome and initiates lytic replication. This is known as spontaneous induction of prophage.

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A lysogenic bacterium is resistant to reinfection by the same or related phages. This is known as superinfection immunity.

Temperate phages are commonly found in clini­cal isolates of gram-positive and gram-negative bacteria and in some cases they contribute to the pathogenicity of the bacteria.

The prophage confers certain new properties on the lysogenic bacterium. This is known as lysogenic conversion or phage conversion.

i. Toxin production in Corynebacterium diphtheriae is determined by the presence of the prophage b in it.

ii. Clostridium botulinum types C and D produce toxin only if these are infected with phage CE b and DE b respectively.

iii. Temperate phages of Salmonella modify the antigenic properties of somatic O antigen in Salmonella.

Importance of Bacteriophages

1. They play an important role in the transmission of genetic information from one bacterium to another by the process of transduction, play a role in the evolution of bacterial types and virulence.

3. Phages may be effective in treating bacterial infections, especially the antibiotic-resistant bacteria, known as Phage therapy.

4. Phages are used as cloning vectors in genetic manipulations and for phage typing to discriminate between bacterial strains.

5. They have a role in the control of bacterial populations in natural waters.

 

Testing of Disinfectants - Evaluation of Antimicrobial Agent Effectiveness

Chemical antimicrobial agents or Disinfectants used in hospitals and laboratories must be periodically tested to determine its efficacy.

Disinfection Process Validation is defined as establishing documented evidence that a disinfection process will consistently remove or inactivate known or possible pathogens from inanimate objects.  Testing of antimicrobial agents is regulated by two different federal agencies. The U.S. Environmental Protection Agency regulates disinfectants, whereas agents used on humans and animals are under the control of the Food and Drug Administration. 

There are several methods of testing disinfectants, and each method have advantages and disadvantages.  These tests are divided into the following disinfectant tests: carrier test, suspension test, capacity test, practical test, field test or in-use test.

Carrier tests

These tests are the oldest tests and was described by Robert Koch in 1881.  In this test, a carrier such as a silk or catgut thread or a penicylinder (a little stick) is used.  This carrier is contaminated by submersion into a liquid culture of the test organism.  The test organism used by Robert Koch was liquid culture of Bacillus anthracis and he used a silk thread.  The carrier will be then dried and brought in contact with the disinfectant to be tested for a given exposure time. After the exposure, the carrier is cultured in a nutrient broth; no growth indicates activity of the disinfectant tested whereas growth indicates that it is not effective as a disinfectant.   Example of a carrier test is the use-dilution test.

Limitation of the carrier tests are: 

a) the number of bacteria dried on a carrier cannot be standardized 

b) the survival of the bacteria on the carrier during drying is never constant.

Suspension tests

In these tests, a sample of the bacterial culture is suspended into the disinfectant solution and after exposure it is verified by subculture whether this inoculum is killed or not. Suspension tests are preferred to carrier tests as the bacteria are uniformly exposed to the disinfectant. There are different kinds of suspension tests, they are the qualitative suspension tests, quantitative suspension tests and the phenol coefficient test.

In a qualitative suspension test, a loopful of bacterial suspension will be brought into contact with the disinfectant and then a loopful of this mixture will be cultured for surviving organisms. Results were expressed as ‘growth’ or ‘no growth’. 

In quantitative suspension test, the same above procedure will be carried out, but here the number of surviving organisms are counted and compared to the original inoculum size.

Phenol coefficient test: Phenol coefficient of a disinfectant is calculated by dividing the dilution of test disinfectant by the dilution of phenol that disinfects under predetermined conditions.

1.       Rideal Walker method:  Phenol will be diluted to different concentrations and the test disinfectant also will be diluted to different concentrations. Their bactericidal activity will be determined against Salmonella typhi suspension. Subcultures are performed from both the test and phenol at various time intervals of 2.5, 5, 7.5 and 10 minutes. The plates are incubated for 48-72 hours at 37°C. That dilution of disinfectant which disinfects the suspension in a given time is divided by that dilution of phenol which disinfects the suspension in same time gives its phenol coefficient. Suppose that the phenol dilution was 1/90 and maximum effective dilution for disinfectant was 1/450. The phenol coefficient of disinfectant would be 5. 

Disadvantages of the Rideal-Walker test are: No organic matter is included; the microorganism Salmonella typhi may not be appropriate; the time allowed for disinfection is short; it should be used to evaluate phenolic type disinfectants only.

2.       Chick Martin test: This test also determines the phenol coefficient of the test disinfectant. Unlike in Rideal Walker method where the test is carried out in water, the disinfectants are made to act in the presence 3% dried human feces to simulate the presence or organic matter. Time for subculture is fixed at 30 minutes and the organism used to test efficacy is S.typhi as well as S.aureus.

3. Garrod’s Test: This test is a modified Chick Martin Test, where the organic material added is 5% of yeast suspension.

Capacity tests

The ability to retain activity in the presence of an increasing load of dirt and bacteria is the capacity of the disinfectant. In a capacity test, the disinfectant is challenged repeatedly by successive additions of bacterial suspension until its capacity to kill has been exhausted. The best known capacity test is the Kelsey-Sykes test.

Kelsey-Sykes test: This is a triple challenge test, and is done to evaluate effectiveness of disinfectant both at clean and dirty conditions.  The dilutions of the disinfectant are made in hard water for testing under clean conditions and in yeast suspension for testing under dirty conditions.  Test organism may be Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris or E coli.  There will be three successive additions of the test bacteria, at 0, 10 and 20 minutes’ interval.  The disinfectant will be evaluated for its ability to kill the bacteria 8 minute after each challenge or addition of test bacteria by inoculating into a nutrient media and bacterial growth if any will be monitored after incubation.   The disinfectant passes at the dilution tested if negative results (no bacterial growth) are obtained after first and second challenge.   The result will be reported as a Pass or Fail, not as a coefficient. 

Stability Test: if the disinfectant found to be effective after capacity test, its stability upon storage should be determined.  So if the prepared disinfectant solutions are to be kept for more than 24 hours, then its stability is measured by using the supplementary test.  Pseudomonas aeruginosa is used.  For this test the disinfectant solution is prepared in two sets

1. First set will be inoculated with the test organism and will be incubated for 7 days and will be tested for bacterial growth after 7 days.

2. The second set will be kept aside at room temperature for 7 days and then inoculated with the test organism and will be tested for bacterial growth.

If bacteria is found to survive the particular dilution of the disinfectant tested, a higher concentration of the disinfectant must be tested. 

Practical tests

The practical tests under real-life conditions are performed after measuring the time-concentration relationship of the disinfectant in a quantitative suspension test. The objective is to verify whether the proposed use dilution is still adequate in the conditions under which it would be used. The best known practical tests are the surface disinfection tests.

Surface disinfection tests assess the effectiveness of the selected sanitizer against surface-adhered microorganisms. The test surface (a small tile, a microscopic slide, a piece of PVC, a stainless steel disc, etc.) is contaminated with a standardized inoculum of the test bacteria and dried: then a definite volume of the disinfectant solution is distributed over the carrier; after the given exposure time the number of survivors is determined.

There is an essential difference between a carrier test and a surface disinfectant test.  In the carrier test, the carrier is submerged in the disinfectant solution during the whole exposure time, whereas in the surface disinfectant test, the disinfectant is applied on the carrier for the application time and then the carrier continues to dry during the exposure time. Surface tests can reflect in-use conditions like contact times, temperatures, use-dilutions, and surface properties.

Surface Time kill Test: A 24-hour culture in nutrient broth culture is prepared and spread onto the center of a number of sterile test surfaces. The inoculated test surfaces are treated with the disinfectant, each for different durations of time. After the treatment times, the test surfaces are placed into a solution that neutralizes the disinfecting action, and microorganisms surviving are cultured and enumerated.  Untreated, inoculated test surfaces are kept as control.

In-use test

This was described by Maurer in 1985 to detect contamination of disinfectants. A 1 ml sample of the disinfectant is added to 9 ml diluent that contains an inactivator. Ten drops of this diluted disinfectant are placed on two nutrient agar plates. One is incubated at 37oC for three days and the other at room temperature for seven days. Five or more colonies on either plate indicate contamination of the disinfectant.

Testing scheme

The antimicrobial efficiency of a disinfectant is examined at three stages of testing. First phase involves laboratory tests which verify whether the chemical compound possesses antimicrobial activity, generally by quantitative suspension tests are considered. Second stage is carried out in laboratory using conditions simulating real-life conditions. Here disinfection procedures are examined by performing practical tests.  Third phase involves field tests and in-use tests.

The antimicrobial activity is assessed in terms of activity towards vegetative bacteria (bactericidal tests), against fungi and yeasts (fungicidal tests), against mycobacteria (tuberculocidal tests), against viruses (virucidal tests) and against spores of bacteria (sporicidal tests).