Describe bacterial genetics including the mechanisms of gene transfer and the basis of drug resistance.

Bacterial Genome and Variation

• The bacterial genome is a single circular double-stranded DNA molecule with no nuclear membrane and no introns; it is haploid, so any mutation is immediately expressed
• Extrachromosomal elements — plasmids (autonomous circular DNA, often carrying resistance or virulence genes; the F factor confers the ability to conjugate); transposons ("jumping genes" that move between DNA molecules and can carry resistance genes); and integrons, which capture and express gene cassettes and are a major vehicle for multidrug resistance
• Variation arises by mutation or by gene transfer, and the distinction matters clinically: mutation is vertical and slow, gene transfer is horizontal and can spread resistance between species overnight

Mechanisms of Gene Transfer

Mechanisms of Drug Resistance

Genetic Techniques in Diagnosis

• Polymerase chain reaction (PCR) — amplifies a target sequence; rapid, extremely sensitive, and detects organisms that are non-cultivable, slow-growing or already suppressed by antibiotics. Real-time PCR quantifies, as in HIV and hepatitis viral load
• CBNAAT / GeneXpert — a cartridge-based automated PCR that detects Mycobacterium tuberculosis and rifampicin resistance within about 2 hours; it has transformed tuberculosis diagnosis in India
• Line probe assay — detects mutations conferring resistance to isoniazid, rifampicin and second-line drugs
• Nucleic acid hybridisation and DNA probes; whole genome sequencing for outbreak investigation and resistance prediction
• Limitations of molecular methods — they detect nucleic acid, not viability, so they may remain positive after successful treatment; they are prone to contamination giving false positives; and they do not provide an isolate for full sensitivity testing

Bacterial Mutation and its Consequences

• Mutation is a spontaneous heritable change in the base sequence, occurring at a rate of roughly 10-6 to 10-9 per cell division; it is random and not induced by the drug
• Because bacteria are haploid, a mutation is expressed immediately, without the masking that a second allele would provide
• Types — point mutation (substitution), frameshift (insertion or deletion), and deletion of larger segments
• Clinical consequences — single-step high-level resistance to rifampicin (rpoB), streptomycin and fluoroquinolones (gyrA), which is exactly why these drugs must never be used alone in tuberculosis
• The mathematics of combination therapy — if resistance to one drug arises at 10-6 and to another at 10-8, an organism resistant to both arises at 10-14, which exceeds the number of bacilli in any lesion. This is the whole rationale for multidrug regimens in tuberculosis, leprosy and HIV
• Phenotypic variation — changes in expression without genetic change, such as capsule loss on subculture or the smooth-to-rough colony transition

Genetic Basis of Vaccines and Diagnostics

• Recombinant vaccines — the gene for a protective antigen is expressed in yeast or another host: the hepatitis B surface antigen vaccine and the HPV vaccine are made this way, avoiding any need to handle the pathogen
• Live attenuated organisms are produced by repeated subculture or by deliberate deletion of virulence genes, which gives a more stable attenuation than the classical method
• MRNA and viral vector vaccines deliver the genetic instructions for an antigen rather than the antigen itself, and the platform can be redirected to a new pathogen very quickly — as the COVID-19 vaccines demonstrated
• Molecular diagnosis — PCR and real-time PCR, CBNAAT detecting M. Tuberculosis and rifampicin resistance within about 2 hours, line probe assays, and whole genome sequencing for outbreak tracing and resistance prediction
• Molecular typing — pulsed-field gel electrophoresis, multilocus sequence typing and sequencing have largely replaced phage typing where they are affordable, and allow an outbreak strain to be traced between wards, hospitals and countries
• CRISPR was discovered as a bacterial immune system against phages, storing fragments of previous invaders to recognise them again; it is now the basis of genome editing — a reminder that fundamental microbiology repeatedly yields tools nobody was looking for
• Genetically engineered organisms produce many therapeutic proteins — human insulin, growth hormone, erythropoietin, interferons and clotting factors — which removed the risks of animal and cadaveric sources
• Antibiotic resistance genes are used as markers in genetic engineering, which has itself raised concern about their environmental spread
• Resistance carries a fitness cost in most organisms, so reducing antibiotic use allows sensitive strains to re-establish; resistance is therefore reversible in principle, which is the strongest argument for stewardship
• The gut is the great mixing vessel — commensals and pathogens exchange plasmids there under the selective pressure of whatever antibiotic the patient is taking, which is why an antibiotic given for a chest infection can select urinary resistance
• Environmental and agricultural use contributes substantially, and resistant organisms reach humans through food, water and effluent; this is a public health problem beyond clinical prescribing
• Never add a single drug to a failing regimen — that is functional monotherapy against an already-resistant population, and resistance to the added drug follows within weeks
• Fixed-dose combinations prevent inadvertent monotherapy, which is why they are used in tuberculosis, leprosy and HIV programmes where supervision cannot be guaranteed
• Detecting resistance genes is faster than growing the organism, and CBNAAT reporting rifampicin resistance in 2 hours has changed the management of tuberculosis in India more than any new drug
• A gene may be present without being expressed, so molecular resistance testing complements rather than replaces culture and phenotypic sensitivity

Applied Aspects

• Antibiotics do not create resistance; they select for it — resistant mutants arise spontaneously, and the drug kills the sensitive majority and leaves them to multiply without competition
• Conjugation is why resistance spreads so fast — a plasmid carrying several resistance genes can move between species in the gut of a single patient, so using one antibiotic can select resistance to others
• Combination therapy prevents resistance in tuberculosis, leprosy and HIV because the probability of simultaneous resistance to several drugs is the product of the individual probabilities
• Molecular detection of rifampicin resistance guides treatment within hours instead of weeks, which is the single most useful diagnostic advance in Indian tuberculosis practice
• Infection control limits the spread of resistant organisms, and no prescribing policy compensates for poor hand hygiene; the two must go together
• India has among the highest rates of antimicrobial resistance in the world, driven by over-the-counter sale, incomplete courses and agricultural use; the National Action Plan and Schedule H1 are steps toward control