Bacteriology
1. What this chapter covers, and how NEET PG actually tests it
Bacteriology looks like a catalogue of organisms, each with its own list of properties.
It is better approached as a decision tree, because the laboratory identifies organisms by a fixed sequence and the exam follows that same sequence.
Gram stain first, then shape and arrangement, then one distinguishing test. Three steps narrow almost any organism to a small group before clinical features are considered at all.
| Step | What it gives you |
|---|---|
| Gram reaction | Splits the entire field in two |
| Shape and arrangement | Cocci in clusters, chains, pairs; bacilli |
| One key test | Catalase, coagulase, oxidase, lactose fermentation |
| Growth requirement | Special media, atmosphere, temperature |
| Toxin or virulence factor | Explains the clinical syndrome |
The Gram reaction reflects cell wall structure: Gram-positive organisms have thick peptidoglycan retaining crystal violet, while Gram-negative organisms have thin peptidoglycan and an outer membrane containing lipopolysaccharide.
Lipopolysaccharide is why Gram-negative sepsis produces such profound shock, since its lipid A component is a potent trigger of cytokine release.
2. Gram-positive cocci
2.1 The catalase and coagulase branch
Catalase separates staphylococci, which are positive, from streptococci and enterococci, which are negative.
Coagulase then separates Staphylococcus aureus, which is positive, from the coagulase-negative staphylococci.
Those two tests, in that order, identify the commonest Gram-positive pathogens in two steps.
Staphylococcus epidermidis colonises prosthetic material and forms biofilm, while Staphylococcus saprophyticus causes urinary infection in young women and is novobiocin-resistant.
2.2 Staphylococcus aureus and its toxins
Many of its syndromes are toxin-mediated rather than invasive, which is why some occur without the organism being present at the site of illness.
Toxic shock syndrome toxin acts as a superantigen, cross-linking the T cell receptor to MHC class II outside the antigen-binding groove.
That mechanism activates a huge fraction of T cells simultaneously, producing massive cytokine release, and it explains why the illness is so fulminant.
Exfoliative toxin causes scalded skin syndrome by cleaving desmoglein, and enterotoxin causes food poisoning with vomiting within one to six hours because it is preformed in the food.
The short incubation is the diagnostic clue, since an organism that has to multiply first would take much longer.
2.3 Streptococci
Haemolysis on blood agar divides them: beta-haemolytic organisms lyse red cells completely, alpha-haemolytic produce green discolouration, gamma produce none.
| Organism | Group | Distinguishing test |
|---|---|---|
| Streptococcus pyogenes | A, beta | Bacitracin sensitive |
| Streptococcus agalactiae | B, beta | CAMP positive, hippurate positive |
| Streptococcus pneumoniae | Alpha | Optochin sensitive, bile soluble |
| Viridans streptococci | Alpha | Optochin resistant |
| Enterococcus | D | Growth in 6.5 per cent salt and bile |
Streptococcus pyogenes causes both suppurative disease and two non-suppurative sequelae: rheumatic fever through molecular mimicry, and post-streptococcal glomerulonephritis through immune complex deposition.
Only rheumatic fever is prevented by treating the pharyngitis, since glomerulonephritis follows skin as well as throat infection and antibiotic treatment does not reliably prevent it.
Group B streptococcus is the leading cause of neonatal sepsis, which is why maternal screening and intrapartum prophylaxis exist.
2.4 The Gram-positive bacilli
These are fewer in number and each is identified by a single memorable feature.
Corynebacterium diphtheriae grows on tellurite and Loeffler medium and produces a toxin inhibiting elongation factor 2, which halts protein synthesis.
The pseudomembrane in diphtheria is dangerous mechanically as well as toxically, since it can obstruct the airway, and the toxin additionally causes myocarditis and neuritis.
The Elek test demonstrates toxin production, which matters because non-toxigenic strains exist and cause much milder disease.
Listeria monocytogenes shows tumbling motility at 22 degrees, grows at refrigeration temperature, and crosses the placenta, which is why it causes neonatal and pregnancy-associated infection.
It is intrinsically resistant to cephalosporins, which is exactly why ampicillin is added when listeria is a possibility in meningitis.
Bacillus anthracis is non-motile with a polypeptide capsule and produces a black eschar in cutaneous disease, while Bacillus cereus causes the two food poisoning syndromes.
Nocardia is partially acid-fast, aerobic and branching, whereas Actinomyces is anaerobic, not acid-fast, and produces sulphur granules.
3. Gram-negative organisms
3.1 The enterics
Lactose fermentation on MacConkey agar is the first division: Escherichia coli, Klebsiella and Enterobacter ferment lactose; Salmonella, Shigella and Proteus do not.
Oxidase separates Pseudomonas, which is positive, from the Enterobacteriaceae, which are negative.
| Organism | Key feature |
|---|---|
| Escherichia coli | Commonest cause of urinary infection and Gram-negative sepsis |
| Klebsiella | Currant-jelly sputum, alcoholics, mucoid capsule |
| Proteus | Swarming growth, urease, struvite stones |
| Salmonella typhi | Sustained fever, relative bradycardia, rose spots |
| Shigella | Very low infective dose, bloody diarrhoea |
| Vibrio cholerae | Rice-water stools, comma-shaped, oxidase positive |
| Pseudomonas aeruginosa | Blue-green pigment, grape-like odour, oxidase positive |
Shigella needs only a handful of organisms to cause disease, which is why person-to-person spread is so efficient, whereas Salmonella requires a far larger inoculum and is usually food-borne.
Cholera toxin permanently activates the Gs protein, locking adenylate cyclase on, and the resulting cyclic AMP drives massive chloride and water secretion.
That is why cholera produces watery stools without inflammation or blood, and why oral rehydration containing glucose works, since glucose-coupled sodium absorption remains intact.
3.2 The diarrhoeal Escherichia coli
Five pathotypes are distinguished by mechanism rather than by appearance.
Enterotoxigenic strains cause traveller's diarrhoea through heat-labile and heat-stable toxins, the labile toxin working like cholera toxin.
Enterohaemorrhagic strains, particularly O157:H7, produce Shiga toxin, cause bloody diarrhoea without fever, and can lead to haemolytic uraemic syndrome.
Antibiotics are avoided in O157:H7 because killing the organism releases more toxin and increases the risk of haemolytic uraemic syndrome.
Enteroinvasive strains resemble Shigella, enteropathogenic strains cause infantile diarrhoea by attaching and effacing microvilli, and enteroaggregative strains cause persistent diarrhoea.
3.3 The fastidious and atypical organisms
Haemophilus influenzae requires factors X and V, which is why it grows on chocolate agar but not blood agar.
Bordetella pertussis produces a toxin that inhibits Gi, the mirror image of cholera toxin's action on Gs, and both raise cyclic AMP by opposite routes.
Legionella grows on buffered charcoal yeast extract, causes pneumonia with hyponatraemia and diarrhoea, and is diagnosed by urinary antigen.
Mycoplasma has no cell wall, is therefore resistant to all beta-lactams, and causes atypical pneumonia with cold agglutinins.
Chlamydia is an obligate intracellular organism with a two-form life cycle: the elementary body infects and the reticulate body replicates.
The elementary body is the extracellular infectious form and the reticulate body the intracellular replicating one, which is why the organism cannot be cultured on ordinary media and why treatment requires an agent penetrating cells.
Chlamydia trachomatis serovars determine the disease: A to C cause trachoma, D to K cause genital infection and neonatal conjunctivitis, and L1 to L3 cause lymphogranuloma venereum.
Rickettsiae are also obligate intracellular organisms, transmitted by arthropods, and cause the typhus and spotted fever groups.
The Weil-Felix test exploits cross-reactivity with Proteus antigens and is now largely historical, having been replaced by serology and molecular methods.
3.4 Neisseria and the zoonoses
Both Neisseria species are oxidase-positive Gram-negative diplococci, and sugar fermentation separates them: meningococcus ferments maltose and glucose, gonococcus only glucose.
Only meningococcus has a polysaccharide capsule, which is why a vaccine exists for it and not for gonococcus, and why terminal complement deficiency predisposes specifically to neisserial disease.
Gonococcus undergoes rapid antigenic variation of its pili, which is why natural infection confers no immunity and why reinfection is common.
Brucella causes undulant fever with a history of unpasteurised dairy or animal contact, and is a recognised laboratory-acquired infection.
Yersinia pestis causes plague with buboes and shows bipolar safety-pin staining.
Bartonella causes cat-scratch disease, and Pasteurella multocida causes rapidly developing cellulitis after a cat or dog bite, often within hours.
The speed of onset after a bite is the discriminating feature, since staphylococcal or streptococcal wound infection takes considerably longer.
4. Mycobacteria, anaerobes and spirochaetes
4.1 Mycobacteria
The cell wall is rich in mycolic acid, which makes the organism acid-fast and accounts for its slow growth and environmental resilience.
Ziehl-Neelsen staining exploits that property, and fluorescent auramine staining is more sensitive for screening.
Cartridge-based nucleic acid amplification testing detects both Mycobacterium tuberculosis and rifampicin resistance in about two hours, which is why it has replaced smear microscopy as the initial test under India's programme.
Culture on Lowenstein-Jensen medium remains the reference standard but takes weeks, which is precisely the delay the molecular test avoids.
Smear microscopy detects only around ten thousand bacilli per millilitre, which is why smear-negative disease is common and why a negative smear never excludes tuberculosis.
Non-tuberculous mycobacteria are distinguished by their growth characteristics: Mycobacterium avium complex causes disseminated disease in advanced HIV, and Mycobacterium marinum causes swimming pool granuloma.
Mycobacterium leprae cannot be cultured on artificial media at all, and is classified by the Ridley-Jopling scale from tuberculoid, with few organisms and strong immunity, to lepromatous, with abundant organisms and weak cell-mediated immunity.
The lepromin test is positive in tuberculoid and negative in lepromatous disease, which reflects that immunity gradient rather than the burden of organisms.
4.2 Anaerobes and spore-formers
Clostridium tetani produces tetanospasmin, which blocks release of glycine and GABA from inhibitory interneurons, causing unopposed muscle contraction and spastic paralysis.
Clostridium botulinum produces a toxin blocking acetylcholine release at the neuromuscular junction, causing flaccid paralysis.
The two toxins are mechanistically similar cleavers of SNARE proteins, and the opposite clinical pictures come from which neuron they act on, inhibitory in tetanus and motor in botulism.
Clostridium perfringens causes gas gangrene through alpha toxin, a lecithinase that destroys membranes.
Clostridioides difficile causes pseudomembranous colitis through toxins A and B, typically after antibiotic therapy.
All four clostridia are spore-forming anaerobes, and the spore is what allows them to persist in soil, dust and hospital surfaces long after vegetative organisms would have died.
That resilience is why alcohol hand rub does not inactivate Clostridioides difficile spores and why soap and water handwashing is required instead.
Bacteroides fragilis is the dominant non-spore-forming anaerobe of the colon and is a common component of intra-abdominal abscesses.
Anaerobic infections in general are suggested by foul-smelling discharge, gas in tissues, and a site adjacent to a mucosal surface where anaerobes normally reside.
4.3 Spirochaetes
Treponema pallidum cannot be cultured and is diagnosed serologically, using non-treponemal tests such as VDRL and RPR for screening and activity, and treponemal tests for confirmation.
Non-treponemal titres fall after treatment while treponemal tests stay positive for life, which is why the two serve different purposes.
Leptospira causes Weil disease with jaundice and renal failure, and is associated with water exposure and rodent urine.
Borrelia burgdorferi causes Lyme disease with erythema migrans, and Borrelia recurrentis causes relapsing fever through antigenic variation.
The relapses in relapsing fever occur because each wave of antibody clears one antigenic variant while a new one emerges, which is the same evasion strategy the gonococcus uses with its pili.
The Jarisch-Herxheimer reaction follows treatment of any spirochaetal infection, and is caused by sudden release of antigen from killed organisms rather than by drug allergy.
5. Bacterial genetics and sterilisation
5.1 How resistance moves between bacteria
Bacteria acquire new genes by three mechanisms, and distinguishing them is a reliable examination point.
Transformation is uptake of naked DNA from the environment, and only naturally competent organisms such as Streptococcus pneumoniae, Haemophilus and Neisseria do it readily.
Transduction is transfer by a bacteriophage, and it comes in two forms: generalised, where any fragment may be packaged by mistake, and specialised, where genes adjacent to the prophage insertion site are carried.
Diphtheria and botulinum toxins are both encoded by phage genes, which is why non-toxigenic strains exist and why lysogenic conversion matters clinically.
Conjugation is direct transfer through a sex pilus, requires cell contact, and is the principal route by which resistance plasmids spread.
Transposons move genes within and between DNA molecules, and integrons capture and express resistance cassettes, which is how multiple resistances accumulate together.
5.2 Sterilisation and disinfection
Sterilisation destroys all microbial life including spores; disinfection reduces organisms but does not reliably kill spores.
| Method | Conditions | Use |
|---|---|---|
| Autoclave | 121 degrees, 15 psi, 15 minutes | Most reliable general method |
| Hot air oven | 160 degrees for 2 hours | Glassware, oils, powders |
| Ethylene oxide | Gas, prolonged | Heat-sensitive plastics, endoscopes |
| Glutaraldehyde | 2 per cent immersion | Endoscopes, delicate instruments |
| Pasteurisation | 63 degrees for 30 minutes or 72 for 15 seconds | Milk; does not sterilise |
| Filtration | 0.22 micrometre | Heat-labile fluids, does not remove viruses |
Moist heat kills faster than dry heat at the same temperature because it denatures proteins rather than merely oxidising them, which is why the autoclave works at 121 degrees while the hot air oven needs 160.
Spore-forming organisms are the benchmark for sterilisation, and Geobacillus stearothermophilus spores are used to validate autoclave cycles.
Prions resist all conventional methods and require extended autoclaving with sodium hydroxide.
6. Worked examples
Example 1
A child develops vomiting two hours after eating rice from a buffet. Others who ate the same dish are also affected.
The interval is the decisive detail, because two hours is far too short for an organism to multiply and invade.
A very short incubation means a preformed toxin was already in the food.
Staphylococcus aureus enterotoxin and Bacillus cereus emetic toxin both act this way, and the association with reheated rice points specifically to Bacillus cereus.
Illness beginning after twelve hours or more would instead suggest an organism that had to multiply in the gut.
Example 2
A patient with bloody diarrhoea and no fever develops acute kidney injury and thrombocytopenia. Stool culture grows a sorbitol-non-fermenting Escherichia coli.
Sorbitol non-fermentation is the screening characteristic of O157:H7 on selective media.
The organism produces Shiga toxin, which damages endothelium and causes haemolytic uraemic syndrome.
Antibiotics must be avoided, because killing the organism releases stored toxin and increases the risk of haemolytic uraemic syndrome.
Management is supportive, with attention to fluid balance and renal replacement if required, and antimotility agents are also avoided.
Example 3
A patient has trismus and generalised muscle spasms with a clear sensorium following a puncture wound.
Preserved consciousness alongside severe spasm points away from a central nervous system infection.
Tetanospasmin travels retrogradely up the motor neuron and blocks release of glycine and GABA from inhibitory interneurons in the spinal cord.
Removing inhibition leaves motor neurons firing unopposed, which produces spasm without any loss of consciousness.
Botulinum toxin cleaves the same class of protein but acts at the motor terminal itself, so it produces flaccid paralysis instead.
7. Traps the exam sets repeatedly
Giving antibiotics for Escherichia coli O157:H7. Bacterial killing releases toxin and increases the risk of haemolytic uraemic syndrome.
Expecting antibiotic treatment of pharyngitis to prevent glomerulonephritis. It prevents rheumatic fever, not glomerulonephritis.
Using a beta-lactam for mycoplasma. It has no cell wall, so the entire class is useless.
Assuming a positive treponemal test means active syphilis. It stays positive for life; non-treponemal titres indicate activity.
Reading a negative lepromin test as absence of leprosy. It is negative in lepromatous disease, where the organism burden is highest.
Using a cephalosporin where listeria is possible. Listeria is intrinsically cephalosporin-resistant, so ampicillin must be added.
Assuming pasteurisation sterilises. It reduces pathogen load but leaves spores and some organisms viable.
Treating all diphtheria isolates as toxigenic. Non-toxigenic strains exist, which is what the Elek test distinguishes.
Summary
Bacteriology is a decision tree, and the laboratory sequence of Gram stain, morphology and one key test is the sequence the exam follows.
Lipopolysaccharide in the Gram-negative outer membrane is why Gram-negative sepsis causes such profound shock.
Catalase then coagulase identifies the commonest Gram-positive pathogens in two steps.
A short incubation in food poisoning means preformed toxin rather than bacterial multiplication.
Superantigens activate T cells outside the antigen-binding groove, which is why toxic shock is so fulminant.
Rheumatic fever is prevented by treating pharyngitis; post-streptococcal glomerulonephritis is not.
Cholera toxin locks Gs on and pertussis toxin inhibits Gi, raising cyclic AMP by opposite routes.
Antibiotics are avoided in O157:H7 because killing the organism releases Shiga toxin.
Tetanus and botulinum toxins both cleave SNARE proteins, and the opposite pictures reflect which neuron is affected.
Non-treponemal serology tracks disease activity while treponemal tests remain positive for life.
Resistance moves between bacteria by transformation, transduction and conjugation, with conjugation carrying most resistance plasmids.
Diphtheria and botulinum toxins are phage-encoded, which is why non-toxigenic strains exist.
Moist heat kills faster than dry heat because it denatures rather than oxidises, which is why the autoclave works at a lower temperature than the hot air oven.
