Selective toxicity, Minimal inhibitory concentration and Minimal lethal concentration

Selective toxicity: Antimicrobial drugs must have selective toxicity. Selective toxicity means the ability of a drug to kill or inhibit the microbial growth with minimal or no damage to the host cell.

Minimal inhibitory concentration (MIC): It is defined as the lowest concentration of an antimicrobial drug that inhibits the visible growth of microbes after overnight incubation. It can be determined by preparing a solution of a drug at increasing concentration and then incubating it with separate batches of cultured microorganism. After incubation, the result is measured either by agar or broth dilution method.

Minimal lethal concentration (MLC): It is defined as the lowest concentration of antimicrobial drug that kills a particular microorganism.

Broad-spectrum antibiotic and narrow-spectrum antibiotic

On the basis of target specificity, antibiotics are classified into two types:

Broad-spectrum antibiotic: Those antibiotics that attack a wide range of pathogens are called broad-spectrum antibiotic.

Advantages:

They are beneficial when an infection is caused by multiple groups of bacteria or when infection is suspected but the causative organism is unknown.

Limitations:

As they attack many different kinds of pathogens, they kill normal bacteria also. They also aid in the development of antimicrobial resistance. Due to the continuous exposure to the same antibiotic, microbes may develop changes in their structure or function and becomes resistant to the antibiotic.

Examples:

Ciprofloxacin, Ampicillin, Rifampin, Tetracyclines, Chloramphenicol, Amoxicillin, Ticarcillin etc.

Narrow-spectrum antibiotic: They are effective against a specific organism. They are helpful when a known organism causes infection.

Advantages:

As they are effective against a particular organism, they will not kill the normal microorganisms of host body. So, it has less ability to cause superinfection. Also, they will develop less antimicrobial resistance.

Limitations:

They can be used only if causative agent is known.

Examples:

Bacitracin, Dapsone, Gentamicin, Methicillin, Clindamycin, Vancomycin, Azithromycin etc.

Therapeutic Index

The therapeutic index (TI) is the ratio of toxic dose to the therapeutic dose. A chemotherapeutic agent that is used for the treatment must have a toxic dose and a therapeutic dose.

The therapeutic dose is defined as the dose of a drug that is required for the clinical treatment of a particular infection. Whereas, toxic dose is defined as the dose of a drug at which it becomes toxic for the host.

A drug, used for the treatment, must have a toxic effect on organism but not on the host. The larger the TI, the safer the drug will be. A higher TI indicates a significant difference between toxic dose and therapeutic dose. With higher TI, a patient would have to take a much higher dose of the drug to reach the toxic level as compared to the dose required in eliciting the therapeutic effect. If the TI is small, it means the difference between the toxic dose and therapeutic dose is very small. In such cases, the drug needs to be monitored very carefully, as a minor increase in the dose of the drug can make it toxic from therapeutic.

For example, penicillin inhibits bacterial cell wall synthesis and as host cell lacks cell wall, so penicillin does not have an effect on host cell. This indicates that penicillin has high TI. But, if the drug inhibits the same process in the host cell also, it is considered to have low TI.

TI has certain limitations as it is not possible to measure the toxic dose of a drug in humans. Although animal studies help in measuring the toxic dose, it can’t be fully accepted for humans. Nevertheless, TI highlights the significance of the margin of safety, as distinct from the potency, in determining the usefulness of a drug.

Plasmid

In 1952, an American molecular biologist, Joshua Lederberg, first introduced the term plasmid. Plasmids are double-stranded DNA molecules, mostly circular, negatively supercoiled that can exist and replicate independent of the main chromosome or may get integrated into the main chromosome. Basically, they are autonomous, self-replicating molecules of DNA that are present as an extra-chromosomal genetic material in bacteria. They are generally DNA molecules; RNA plasmids are rare. They are mostly found in bacteria, but sometimes they are present in archaea and eukaryotic organisms also. They range in size from less than 5 bp (base pair) to over 1000 kbp (kilo-base pair). They are usually not attached to the plasma membrane and sometimes are lost to one of the progeny cells during cell division i.e. the division of plasmids to daughters cells is not always equal. Also, multiple plasmids can coexist in the same cell, each with a different function.

Plasmids are very small and they are not required for the normal growth and reproduction. They carry genes that provide bacteria with some selective advantage like drug resistance, new metabolic activities or making them pathogenic.

Types of Plasmid:

Conjugative plasmids: A set of transfer (tra) genes that helps in the sexual conjugation between different cells are present in these plasmids. These plasmids help bacteria to transfer plasmids from one bacteria to another by the process of conjugation i.e. via sex pili which is encoded by some tra genes.

Mobilizable plasmids: Tra genes are absent in these plasmids but they contain an oriT site. They are transferred from one bacteria to another by a self-transmissible plasmid. Such process of transfer is known as mobilization and such plasmids are called mobilizable plasmids.

Self-transmissible plasmids: They encodes all the proteins that are required for the transfer between donor and recipient i.e they are both mobilizable and conjugative.

Non-transmissible plasmids: They lack the genes necessary for effective contact and DNA transfer.

On the basis of functions, plasmids may be classified into five groups:

Fertility plasmids (F-plasmids): The F plasmid generally contains the gene that allows DNA transfer between cells. They are commonly found in E.coli. Cells that possess a copy of F-plasmid are called F-plus (F⁺) and cells that lack are called F-minus or F-negative (F^-). Also, F-plasmids are episome (a plasmid that can exist autonomously or can integrate into bacterial chromosome are known as episome). A bacterial cell having F-plasmid integrated into its chromosome are called Hfr (High frequency recombination) cell. The F⁺ cells have a tube-like structure called a pilus that helps in making contact with F^- cells and thereby helps in DNA transfer by a process called conjugation.

Resistance(R) plasmids: They contains genes that makes host cell resistant to one or more antibiotics. The gene basically codes for the enzymes that can alter or destroy the antibiotics. Some R plasmids have single antibiotic resistance gene whereas some have many. For example, plasmid pUC18 has only ampicillin resistance gene whereas plasmid pBR322 has ampicillin and tetracycline resistance gene.

Col (colicinogenic) plasmids: They contains gene that codes for a protein called bacteriocin that can kill other bacteria. They prevent the growth of susceptible bacterial strains that do not contain a Col plasmid. Bacteriocin produced by Escherichia coli is termed as colicin. There are different types of colicins, each designated by a letter (for example, colicin B). Each type of colicin has a particular mode of inhibition. 

Degradative plasmids: Degradative plasmids help in the breakdown of unusual substances like toluene, xylene and salicylic acid that are uncommon in nature. They contains gene that codes for some specific  enzymes that help in the deradation of such unusual substances.

Virulence plasmids: They are responsible for turning bacteria into a pathogen. Some bacterial species like E.coli and Salmonella enterica contain several virulence plasmids.

Note: It is important to note that a single plasmid can belong to more than one of these functional groups.

Plasmids can be classified on the basis of compatibility:

Compatible plasmids: A microorganism can possess different types of plasmid. But different types of plasmid can coexist in a single bacterial cell only if they are compatible. If two plasmids are incompatible, then one or other will be lost from the cell. The more distinct the two plasmids are, the more compatible they will be. For example, a cell containing two plasmids and they are encoding for different antibiotics. Such plasmids are compatible plasmids and they will exist together.

Incompatible plasmids: Incompatible plasmids have the same replication or partition mechanism and thus they can’t coexist in the same cell. When two plasmids are closely related to each other, they are called incompatible plasmids and they cannot be kept together in a single cell. For example: If two plasmids present in a single cell are encoding for the same antibiotic and have no other function, then they are called incompatible plasmids.

Note:

Cryptic plasmid: A plasmid that confer no identified functions or phenotypic properties to the host cell are called cryptic plasmid. They contain gene for self-replication.

  

Plasmids in eukaryotic organisms

Plasmids are not only limited to prokaryotic organisms. Eukaryotic organisms like yeast also contain plasmids. One of the most studied yeast plasmid is known as the 2μ circle. It is present in the nucleus of most Saccharomyces cerevisiae strains and is 6.3 kb. It is a circular extrachromosomal element. It is maintained at about 50 to 100 copies per haploid genome of the yeast cells. The origin of replication site from where the replication starts is known as ARS sequence (autonomous replication sequence). The 2μ circle is coated with nucleosome and replication is initiated at ARS by host replication enzymes once per cell cycle.

Nucleoid

One of the main features that distinguish a prokaryotic cell from a eukaryotic cell is the absence of a nuclear membrane. In eukaryotic cells, chromosomes are contained within a membrane-delimited organelle known as the nucleus. On the contrary, prokaryotes lack this membrane-delimited nucleus. In prokaryotes, chromosome is located in nucleoid which is an irregularly shaped region. Nucleoid is the central region in the prokaryotic cell that contains the genetic material i.e. DNA and is not surrounded by a membrane. Nucleoid is generally composed of 60% DNA, 30% RNA and 10% protein by weight. Transcription and replication of DNA takes place within nucleoid.

The nucleoid is visible under light microscope; after staining with DNA-specific stains such as the Feulgen or Giemsa stain. Both phase contrast microscopy and fluorescence microscopy have been used to visualize the nucleoid. Electron microscopic studies have revealed that nucleoid is associated with either plasma membrane or mesosome. Membranes are also found attached to the isolated nucleoid. This proves that bacterial DNA is attached to cell membrane and also cell membrane may play some role in the separation of DNA into daughter cells during division.

Growth curve

Bacterial reproduction takes place by binary fission in which two daughter cells are produced from a bacterium. When bacteria are grown in a single medium and in a close culture vessel, it is known as batch culture. The growth of bacteria in a batch culture can be plotted as logarithm of number of viable cells in Y-axis and incubation time in X-axis.

This curve has four distinct phases and they are described in detail below:

Lag Phase: When microorganisms are added to the fresh medium, they do not divide immediately. They require some time to adapt themselves to the new environment and to prepare themselves for division. Hence, it is called lag phase. Although cell division does not take place, they are synthesizing RNA, enzymes and other molecules.

Lag phase is important as it allows microorganisms to prepare themselves for cell division. They replicate their DNA, increases their mass and synthesizes required cofactors.  When organisms are transferred from one medium to another, there may be need of some other enzymes to use that nutrient. They can synthesize that during lag phase. Also, if microorganisms are injured they can recover during this time period.

The duration of lag phase varies depending on the nature of medium and condition of microorganism. The lag phase is of short duration when young, exponential phase culture is transferred from one medium to another of same composition. On the contrary, lag phase will be of longer duration if bacteria are transferred from old culture or one which is refrigerated or transferred into the medium of chemically different composition.

Log Phase: This is also known as exponential phase. During this period, cell divides and doubles in number. Doubling occurs at a constant rate i.e. microorganisms are dividing and doubling in number at regular time interval. And because the rate of growth is constant, it results in a straight line. The slope of the line is the growth rate of organism i.e. the measurement of number of divisions per cell per unit time. However, exponential growth cannot continue for long because over the time period nutrient will deplete and waste will accumulate in the medium and eventually population growth will cease.

Stationary Phase: After exponential phase, stationary phase begins during which the growth curves become horizontal.  This phase is marked by the accumulation of waste products and depletion of nutrients in medium. During this phase, growth rate and death rate are balanced. Some organisms are dividing and some are dying keeping the total number of viable cell a constant.

Death Phase: Bacteria begins to die during this phase. Detrimental environmental condition like toxic waste builds up in the medium and nutrients depletions leads to the decline in the number of viable cells. Like the growth of bacteria during log phase, the death of bacteria is also logarithmic and results in a straight line. Most of the microbial population dies but some resistant cells can survive.

Nucleolus

Nucleolus (plural nucleoli) is the largest and most noticeable structure within the nucleus of a eukaryotic cell. They are composed up of DNA, RNA and proteins. They vary in number from one to many nucleoli in the nucleus. They are complex, not membrane enclosed organelle of a eukaryotic cell that separates granular and fibrillar regions.  They are present in nondividing cells but generally disappears during the beginning of mitosis and then begins to reassemble in telophase around nucleolar organizer.

They are the site of ribosome biogenesis and forms rRNA (ribosomal RNA). The rRNA combines with ribosomal proteins and forms ribosomal subunits. These ribosomal subunits are immature and they leave the nucleus through nuclear envelope pores. Then, they matures in the cytoplasm. Nucleoli also help in the assembly of signal recognition particles, modification of tRNA (transfer RNA) and also play a major role in cell’s response to stress.