This document discusses several topics in bacterial genetics and their clinical applications: 1. It describes mechanisms of inherent and acquired drug resistance in bacteria. Acquired resistance can occur through spontaneous mutation or horizontal gene transfer. 2. Molecular diagnostic methods like nucleic acid probes, PCR, and RFLP can be used to identify and type bacteria. PCR allows detection of small amounts of bacterial nucleic acids. 3. Genetic engineering techniques allow isolation of bacterial genes and production of useful proteins like insulin through recombinant DNA technology.

1. Bacterial genetics-Clinical
applications
Dr. Himanshu Khatri
Email: himanshubkhatri@yahoo.co.in

2. • Genetic basis of drug resistance
• Molecular methods for diagnosis and typing of bacteria
• Genetic engineering (recombinant DNA technology [rDNA]
technology)

3. Types of resistance
• Innate or intrinsic or inherent
• Acquired

4. Inherent drug resistance
• Some species, genus or even groups are inherently resistant to an
antibiotic or a group of antibiotics
• Examples include:
1. Gram positive bacteria are inherently resistant to aztreonam and
colistin
2. Gram negative bacteria are inherently resistant to vancomycin and
linezolid

5. Acquired drug resistance
• Bacteria acquire genes for resistance spontaneously or from other
bacteria
• This is usually due to:
1. Mutation (spontaneous acquisition)
2. Horizontal gene transfer (HGT) (acquisition from other bacteria)

6. Mutational drug resistance
• It is less frequent than drug resistance due to HGT
• It is usually chromosomal
• In any population of bacteria, a small proportion of bacteria undergo
spontaneous mutation
• These mutations can give rise to aberrant genes, which may confer
resistance to an antibiotic, which was previously susceptible
• The genes confer resistance by changing the protein structure
• The new protein can be a modified target site to which the antibiotic
cannot bind, an enzyme which can inactivate the antibiotic, an altered
porin for reduced permeability of the antibiotic, or an efflux pump to
pump out the antibiotic from the bacterial cell

7. Drug resistance-mechanisms

8. Antibiotic selection pressure

9. Antibiotic selection pressure
• In a population of bacteria, some bacteria undergo mutation and
develop resistance to an antibiotic, while the non-mutated bacteria,
which are susceptible to an antibiotic, are the significant population.
• When that particular antibiotic is used, the susceptible bacteria are
killed, and the mutant bacteria become the significant population.
• This phenomenon is called antibiotic selection pressure, as the
antibiotic causes a selective pressure by killing the susceptible
bacteria, allowing the antibiotic-resistant bacteria to survive and
multiply

10. Mutational drug resistance…clinical
applications
• Mutational drug resistance plays an important role in development of
resistance during treatment of HIV and M. tuberculosis
• If HIV or M. tuberculosis are treated with a single drug, there is high
probability of development of mutants resistant to that drug
• However, if they are treated with a combination of drugs, the
probability of emergence of mutants becomes very low
• This is because, if a mutant develops resistance to one drug, it is killed
by the other drugs.
• The probability of development of resistance to multiple drugs
simultaneously is next to impossible.

11. Transferable drug resistance
• In this mechanism of drug resistance, the genes responsible for
resistance are transferred from one bacteria to another
• These genes are usually present in the plasmids, but can also be
present in the transposons
• The most common method of transfer is conjugation, although other
methods like transduction and transformation are possible

12. Transferable drug resistance...clinical
applications
• The most common example of plasmid mediated transferable drug
resistance is the transfer of R factor
• R factor contains multiple genes, each of which confers resistance to
an antibiotic. This makes the organism resistant to several antibiotics
simultaneously
• Hence, treatment with multiple antibiotics to overcome resistance
(like mutational drug resistance), is not useful

13. Emergence of drug resistance…prevention
• The only effective way to prevent emergence of drug resistance is by
reducing the antibiotic selection pressure. This can be achieved by
the following:
1. Antibiotics should be prescribed only when absolutely necessary
2. Broad range antibiotics should not be used, unless clinically
indicated. This reduces the antibiotic pressure on the bacterial
population, and the lowers the probability of emergence of mutants
3. Appropriate dosages of antibiotics should be given for the
appropriate period of treatment

14. Molecular methods for identification and
typing of bacteria
• Nucleic acid probes
• Polymerase Chain Reaction (PCR)
• Restriction Fragment Length Polymorphism (RFLP)

15. Nucleic acid probes

16. Nucleic acid probes
• They are short segments of DNA or RNA which have a sequences
complementary to the specific segments of nucleic acid of various
organisms
• Due to their complementarity, a specific probe hybridizes with the nucleic
acid of a particular organism
• Hybridization can be detected by tagging or ‘labelling’ these probes or the
complimentary target DNAs with molecular markers
• The markers are either radioactive or fluorescent molecules, which can be
visualized by using X-ray or UV light respectively
• These probes can be used directly on the clinical samples or on the culture
isolates
• The nucleic acid of organisms are not amplified, as in the polymerase chain
reaction (PCR)

17. Polymerase Chain Reaction (PCR)
• It is a process, where the nucleic acid of organisms are amplified first,
and then analyzed
• It can detect even minute quantities of nucleic acid, as compared to
nucleic acid probes

20. PCR cycle
• PCR is a cyclic process, consisting of three steps
• The first step of PCR cycle is denaturation. In this step, temperature is
elevated to 94oC. This causes separation of two strands of DNA.
• The next step is primer annealing. The temperature is lowered to 40
to 60oC. In this step, the primers (which are short synthetic
oligonucleotide sequences complementary to the target sequence),
anneal to the target DNA sequence in the sample
• The final step is primer extension. The temperature is elevated to
72oC. The DNA polymerase extends the primers using the
deoxyribonucleoside triphosphates present in the reaction mix

21. PCR-process
• The PCR cycle is repeated multiple times
• The product of the previous cycle becomes the template for the next
cycle
• After each cycle, the quantity of DNA doubles
• Theoretically, 20 cycles can give one million copies, and 30 cycles can
give around one billion copies from a single DNA copy
• The process is nowadays automated, and takes place inside a
thermocycler
• The thermocycler automatically increases and lowers the
temperature for the PCR cycle to occur

22. Multiplex PCR
• In this modification, multiple sets of primers are used, each of which
targets a particular sequence.
• If the sample contains a target DNA to any one of the primers, that
DNA is amplified
• For example, to a cerebrospinal fluid sample, sets of primers targeting
sequences of common organisms causing meningitis like
Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria
meningitidis may be added
• If the sample contains Streptococcus pneumoniae, its DNA is
amplified and detected

23. Real time PCR (quantitative PCR)
• Usually, amplification results in the formation of amplicons (amplified
DNA sequences of target organisms), which have to be further
analyzed by means like hybridization, sequencing etc.
• Using real time PCR, analysis can occur simultaneously during
amplification
• DNA can also be quantified using this type of PCR

24. Uses of PCR
• Identification of organisms, which are uncultivable, fastidious, or
having a long generation time
• Detection of genes of resistance, for example, rpoB gene for
rifampicin resistance in Mycobacterium tuberculosis
• Quantify organisms, which may be useful is assessment of efficacy of
the treatment, for example, HIV viral load estimation during anti-
retroviral therapy

25. DNA sequencing
• It is the process of determining the exact order of nucleotides within
a DNA molecule
• It is the gold standard for analysis of the genome
• It is costly

26. Restriction Fragment Length Polymorphism (RFLP)
• It is one of the many methods of genotyping
of organisms
• DNA strands are clipped at selected sites
called restriction sites, using specific enzymes
called restriction endonucleases
• The DNA is thus broken down into restriction
fragments of varying length
• Electrophoresis of these fragments is carried
out. Larger fragments travel shorter
distances, while smaller fragments travel
longer distances on gel
• Each strain of a species gives a unique pattern
(RFLP pattern) on electrophoresis, which is
different from an unrelated strain
• Thus, different strains of a species can be
typed by RFLP

27. Pulse field gel electrophoresis (PFGE)
• The DNA fragments can be separated by a specialized type of
electrophoresis, called PFGE
• In this type of electrophoresis, the direction of the electrical field is
constantly changed
• Hence, it can separate much larger fragments of DNA as compared to
conventional DNA electrophoresis
• The RFLP pattern is stained, and then photographed. The photograph is
then analyzed by a computer program
• Computer programs determine whether the strains under analysis are
indistinguishable or distinguishable, and even the degree to which they
may be distinguished

28. Genetic engineering (rDNA technology)
• Principle:
1. Genes coding for any desired protein are isolated from
microorganisms or from cells of animals
2. These genes are introduced into suitable microorganisms, in which
they would be functional
3. These genes would produces the desired protein in the cells in
which they are introduced
4. The desired protein is extracted for use

30. Genetic engineering-process
1. The DNA containing the gene of interest is cleaved using specific
restriction endonucleases
2. The gene is incorporated into suitable vectors (or carriers) such as
plasmids or lysogenic bacteriophages, using the enzyme ligase
3. The vectors then insert the gene into the nucleic acid of organisms,
in which the gene can synthesize the desired protein. The
microorganism usually used in E. coli K12

31. Advantages of genetic engineering
• Proteins are produced:
1. In pure form
2. In large quantities
3. At a reduced cost

32. • Examples of proteins produced by genetic engineering:
1. Human insulin
2. Interferons
3. Somatostatin
4. Growth hormone
5. Hepatitis B vaccine

33. Thank you