Genetics Of Antibiotic Resistance Notes

Bacterial Genetics And Antimicrobial Resistance

Bacterial Genetics

Bacterial genetics deals with the study of heredity and variation seen in bacteria. All hereditary characteristics of the bacteria are encoded in their DNA which is present in chromosome as well in extrachromosomal genetic material as plasmid.

Plasmid

Plasmids are the extrachromosomal ds circular DNA molecules that exist in free state in the cytoplasm of bacteria and also found in some yeasts:

• Not essential for life: Bacteria may gain or lose plasmid during their life time.
• Numbers: They may be present singly or in multiple numbers up to > 40 plasmids per cell.
• Capable of replicating independently
• Episome: Plasmid may integrate with chromosomal DNA of bacteria and such plasmids are called episomes.
• Curing: The process of eliminating the plasmids from bacteria is known as curing.

Read And Learn More: Micro Biology And Immunology Notes

Classification of Plasmids

1. Based on ability to perform conjugation:
   • Conjugative plasmids or self-transmissible plasmids
   • Nonconjugative plasmids or nontransmissible plasmids. They cannot transfer themselves.
2. Based on compatibility: Compatible plasmids and Incompatible plasmids. Only compatible plasmids can stay together inside a cell.
3. Based on function: There are five main classes of plasmids:
4. Plasmid as vector: By their ability to transfer DNA from one cell to another, plasmids have become important vectors in genetic engineering.
   Plasmids contain certain sites where genes can be inserted artificially by recombinant DNA technology.
   Such plasmids can be used for protein production, gene therapy, etc.

Horizontal Gene Transfer in Bacteria

Gene transfer in bacteria can be broadly divided into:

• Vertical gene transfer (transmission of genes from parents to offspring)
• Horizontal gene transfer (transmission of genes from one bacterium to another bacterium).
  This occurs by:

Transformation

Transformation is the process of random uptake of free or naked DNA fragments from the surrounding medium by a bacterial cell and incorporation of this molecule into its chromosome in a heritable form.

It has been studied so far only in certain bacteria: Streptococcus, Bacillus, Haemophilus, Neisseria, Acinetobacter, and Pseudomonas.

• The Griffith experiment (1928) on mice using pneumococci strains provided the direct evidence of transformation.

Transduction

Transduction is defined as transmission of a portion of DNA from one bacterium to another by a bacteriophage.

Types of transduction

1. Generalized transduction: It involves transfer of any part of the donor bacterial genome into the recipient bacteria.
2. Restricted or specialized transduction: Here, only a particular genetic segment of the bacterial chromosome that is present adjacent to the phage DNA is transduced.
   Role of transduction

In addition to chromosomal DNA, transduction is also a method of transfer of episomes and plasmids.

• Drug resistance, e.g. plasmid coded penicillin resistance in staphylococci.
• Treatment: As a method of genetic engineering in the treatment of some inborn metabolic defects.

Lysogenic Conversion

During the temperate or lysogenic life cycle, the phage DNA remains integrated with the bacterial chromosome as prophage.

The prophage acts as an additional chromosomal element which encodes for new characters to the daughter cells.

• Imparts toxigenicity to the bacteria: Phage DNA may code for various toxins abbreviated as ABCDE:
  A and C of Streptococcus pyrogenic exotoxin (SPE), Botulinum toxin C and D, Cholera toxin, Diphtheria toxin and E. coli (Verocytotoxin).

Conjugation

Conjugation refers to the transfer of genetic material from one bacterium (donor or male) to another bacterium (recipient or female) by mating with each other and forming the conjugation tube. It was discovered first by Lederberg and Tatum.

• F+ × F– Mating: When the F+ cell (containing a plasmid called F factor or fertility factor) comes close to the F– cell (lacking F factor), the F factor forms conjugation tube, through which the F factor is transmitted to F– cell ultimately making F– cell into F+ cell.
• HFR Conjugation: F factor being a plasmid, it may integrate with bacterial chromosome and behave as episome.
  • Such donor cells are able to transfer chromosomal DNA to recipient cells with high frequency in comparison to F+ cells, therefore, named as Hfr cells (high frequency of recombination).
  • During conjugation of Hfr cell with an F- cell, only few chromosomal genes along with only a part of the F factor get transferred. Hence, F- recipient cells do not become F+ cells.
• F′ Conjugation: The conversion of an F+ cell into an Hfr cell is reversible.
  • When the F factor reverts from the integrated to free-state, it may sometimes carry with it some chromosomal DNA from adjacent site of its attachment. They are named as F’ factor (F prime factor).
  • When F’ cell conjugates with a recipient (F-), it transfers, along with the F factor, the host DNA incorporated with it. The recipient becomes F’ cell. This process is called sexduction.
• Conjugation plays an important role in the transfer of plasmids coding for antibacterial drug resistance [resistance transfer factor (RTF)] and bacteriocin production [Colicinogenic (Col) factor].
• R factor (or the resistance factor) is a plasmid which has two components.
  • Resistance transfer factor (RTF) is the plasmid responsible for conjugational transfer
    (similar to F factor)
  • Resistance determinant (r): Codes for resistance to one drug. An R factor can have several r determinants.

Antimicrobial Resistance

Antimicrobial resistance can be of two types; intrinsic and acquired.

• Intrinsic resistance: It is the innate ability of a bacterium to resist a class of antibiotics
• Acquired resistance: It is the emergence of resistance in bacteria, by acquiring the drug-resistant genes either by—(1) mutational or by (2) transferable drug resistance

Mechanism of Antimicrobial Resistance

Bacteria develop antimicrobial resistance by several mechanisms.

1. Decreased permeability across the cell wall: Certain bacteria modify their cell membrane porin channels; thereby preventing the antimicrobials from entering into the cell.
   This strategy has been observed in many gram-negative bacteria such as Pseudomonas, Enterobacter and Klebsiella species against drugs such as imipenem, aminoglycosides, and quinolones.
2. Efflux pumps: Certain bacteria possess efflux pumps which mediate expulsion of the drug(s) from the cell, soon after their entry; thereby decreasing the intracellular accumulation of drugs. This strategy has been observed in:
   Escherichia coli and other Enterobacteriaceae against tetracyclines, chloramphenicol
   • Staphylococci against macrolides and streptogramins
   • Staphylococcus aureus and Streptococcus pneumoniae against fluoroquinolones.
3. By modification of the antimicrobial target sites within the bacteria: This strategy has been observed in:
   • MRSA (Methicillin-resistant Staphylococcus aureus): (see chapter-3.1 for details).
   • VRE (Vancomycin-resistant Enterococci) (See chapter-3.2 for details)
   • Streptomycin resistance in Mycobacterium tuberculosis: Due to modification of ribosomal proteins or 16SrRNA.
   • Rifampicin resistance in Mycobacterium tuberculosis: Due to mutations in RNA polymerase.
   • Quinolone resistance (seen in S. aureus and S. pneumoniae): Due to mutations in DNA gyrase enzyme.
4. By enzymatic inactivation: Certain bacteria can inactivate the antimicrobial agents by producing various enzymes such as:
   • Aminoglycoside modifying enzymes like (acetyltransferases, adenyltransferases, and phosphotransferases, produced by both gram-negative and gram-positive bacteria):
   • They destroy the structure of aminoglycosides.
   • Chloramphenicol acetyltransferase: It is produced by members of Enterobacteriaceae
   • β lactamase enzyme production.

Beta-Lactamase Enzymes

β-lactamase enzymes are capable of hydrolysing the β-lactam rings (the active site) of β-lactam antibiotics; thereby deactivating their antibacterial properties.

• It is observed in both gram-positive and gram-negative bacteria
• They are plasmid coded, and transferred from one bacterium to other mostly by conjugation,(except in Staphylococcus aureus where they are transferred by transduction). Beta lactamases can be classified into two ways:
• Ambler’s classification (structural or molecular classification)—See table
• Bush Jacoby Medeiros classification or functional (phenotypic)—Advanced and complex classification

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing methods include:

• Disk diffusion methods: Kirby-Bauer disk diffusion method and Stokes disk diffusion method:
  • Mueller-Hinton agar (MHA) is considered as the best medium Lawn culture method is used to inoculate the organism onto MHA
  • Control strains: ATCC (American Type Culture Collection) strains are used
  • Reporting is done according to CLSI (Clinical and Laboratory Standards Institute) guidelines.
• Dilution tests: Broth dilution method and Agar dilution method:
  • Here, the antimicrobial agent is serially diluted, each dilution is tested with the test organism for antimicrobial susceptibility test, and the MIC is calculated.
  • MIC (minimum inhibitory concentration) is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism.
• Epsilometer or E-test: This is a quantitative method detecting MIC by using the principles of both the dilution and diffusion of antibiotics into the medium.
• Automated methods such as VITEK 2, Phoenix System and MicroScan WalkAway system
• Molecular methods (PCR detecting drug resistant genes) e.g. MecA gene for MRSA.