Last Minute Revision: High-Yield Microbiology Concepts for Exams

Understanding Bacterial Structures: Cell Wall and Motility

The bacterial cell wall is a primary structure used for classification.

Gram-positive bacteria have a very thick peptidoglycan layer. Their cell walls also contain teichoic acid (important for attachment) and pentapeptide bridges that cross-link the peptidoglycan layers. This pentapeptide bridge is a key feature seen when zooming into the peptidoglycan structure (Nam-Nag arrangement).

Gram-negative bacteria have a very thin peptidoglycan layer and an outer membrane outside the peptidoglycan. This outer membrane contains lipopolysaccharide (LPS), which has three regions: O antigen (region 1), polysaccharide core (region 2), and lipid A (region 3). Lipid A is the endotoxin responsible for septic shock. There is a space between the cell wall (peptidoglycan) and the cell membrane called the periplasmic space. Some bacteria, like spirochetes, keep their flagella hidden inside this space (endoflagella). Gram-negative bacteria lack teichoic acid and pentapeptide bridges.

Bacterial motility is typically mediated by flagella. Flagella are very thin and best visualized using a dark field microscope. Some bacteria, however, are motile due to other structures:

• Type four pili are important for motility in Neisseria and Pseudomonas.
• Cilia are responsible for motility in Balantidium coli, which is the only ciliated intestinal protozoa.

Different Bacteria Exhibit Distinct Types of Motility

• Tumbling and Differential motility (tumbling at 22-25°C but not 37°C) are shown by Listeria.
• Darting or shooting star motility is seen in Vibrio cholerae and Campylobacter.
• Corkscrew motility is characteristic of Treponema pallidum (causes syphilis).
• Twitching motility is seen in Trichomonas.
• Swarming motility, which appears as concentric circles of growth on agar, is shown by PVCs:
  • P: Proteus
  • V: Vibrio parahaemolyticus, Vibrio alginolyticus (not V. cholerae)
  • C: Clostridium tetani, Bacillus cereus (causes Chinese fried rice food poisoning)
  • S: Serratia
• Falling leaf motility is characteristic of Giardia lamblia.

Bacterial Growth and Drug Resistance

The bacterial growth curve consists of distinct phases when bacteria are grown in a limited environment:

1. Lag phase: The number of bacteria remains the same, but the size increases as bacteria accumulate metabolites. Maximum size is at the end of this phase.
2. Log phase (Exponential phase): Bacteria divide exponentially, leading to a rapid increase in number. The size of bacteria goes down. This is the phase where bacteria are most susceptible to antibiotics.
3. Stationary phase: The number of bacteria plateaus as growth rate equals death rate. This is when bacteria undergo sporulation, and produce toxins, antibiotics, and bacteriocins.
4. Death or Decline phase: The number of viable bacteria decreases.

Drug resistance is a significant challenge.

• Mutational drug resistance arises from a single mutation (e.g., RPOB gene mutation causing rifampin resistance in TB). It is generally easy to treat using drug combinations.
• Transferable drug resistance involves the transfer of resistance transfer factors (RTF) via plasmids, carrying multiple resistance determinants. This allows bacteria to become resistant to several drugs simultaneously ("jackpot"). It is more severe, more difficult to treat, and increases the bacteria's virulence.

Transfer of resistance determinants often occurs via conjugation, which involves the transfer of plasmids (like the F factor or R factor) between bacteria.

• An F+ (male) bacterium (with F factor plasmid) can transfer a copy of its F factor to an F- (female) bacterium through a conjugation tube, making the F- recipient also F+.
• An HFR (High Frequency Recombinant) male bacterium has the F factor integrated into its chromosome. When conjugating with an F- female, it attempts to transfer its chromosome along with the integrated F factor. Often, the conjugation tube breaks mid-transfer, resulting in the F- recipient remaining F-.
• An F prime (F') plasmid is created when the integrated F factor from an HFR cell excises from the chromosome, carrying some adjacent chromosomal genes with it. When an F' cell conjugates with an F- female, it transfers the F' plasmid, and the recipient becomes F'.

Sterilization and Disinfection Methods

Different methods are used to sterilize or disinfect materials, chosen based on the material's properties (e.g., heat sensitivity).

Filtration is used for heat-sensitive liquids, as it does not involve heat.

Dry heat sterilization uses hot air.

• The hot air oven is a common example.
• Standard conditions are 160°C for 2 hours, although variations exist (e.g., 170°C for 1 hour, 180°C for 0.5 hours).
• It is used for sterilizing glassware, cotton swabs, greasy/oily materials, dusting powder, and liquid paraffin.

Moist heat sterilization uses steam or vapor. It is generally considered better than dry heat as vapor provides more heat.

• Below 100°C:
  • Pasteurization is used for milk. Holder method is 63°C for 30 minutes, and Flash method is 72°C for 20 seconds. Flash pasteurization is considered better as it kills Coxiella burnetii (causes Q fever, associated with donut granuloma).
  • Inspeurization involves heating at 80°C for 20 minutes on 3 consecutive days. It is used for media containing egg, such as LJ (Lowenstein-Jensen) media (for TB) and LSS (Laughler serum slope) (for diphtheria), as egg coagulates at higher temperatures.
• At 100°C:
  • Boiling at 100°C for 10-20 minutes kills most vegetative bacteria but not spores.
  • Tindelization (also known as intermittent sterilization) involves heating at 100°C for 120 minutes on 3 consecutive days. This method is effective at killing spores.
• Above 100°C:
  • The autoclave is a common device, working like a pressure cooker. Standard conditions are 121°C at 15 psi pressure for 15 minutes.
  • To kill prions, higher temperatures and longer times are required: 134°C for 1.5 hours.
  • Autoclaves are used for sterilizing most culture media (except LJ and LSS), most sutures (except catgut), and instruments/sharps (sharps should be put in separate pouches in an open state).

Chemical methods are also used for disinfection and sterilization:

• Glutaraldehyde (marketed as Cidex) is used to disinfect scopes like bronoscopes and endoscopes.
• Isopropyl alcohol is used for cleaning stethoscopes.
• Plasma sterilization (using machines like Sterrad) is a low-temperature method (~50°C) that uses hydrogen peroxide in a vacuum. It is used for heat-sensitive items like urethroscopes and arthroscope and produces less toxic products compared to ETO.
• Ethylene oxide (ETO) is a gas sterilization method used for heat-sensitive items like cardiopulmonary machines and ventilators. It is toxic (carcinogenic, flammable).
• 1% Sodium hypochlorite is used for blood spills.

Sterilization controls are used to ensure the effectiveness of sterilization methods. Biological controls use spores of resistant bacteria. Chemical controls use chemicals that change color upon proper sterilization (e.g., Brownie's tube).

• Gas (ETO): Bacillus Globy G (also known as Bacillus subtilis).
• Hot air oven: Bacillus subtilis (or Bacillus atrophaeus if subtilus is not an option), Clostridium tetani.
• Autoclave and Plasma: Bacillus stearothermophilus.
• Radiation: Bacillus pumilus.
• Filtration: Brevundimonas diminuta (or Serratia marcescens for ionized students). Brevundimonas diminuta is used because its size (0.22 micron) is close to common filter pore sizes.

Exploring the World of Fungi (Mycology)

Fungi have a different cell wall composition compared to bacteria. The main components of the fungal cell wall are chitin and beta 1,3 D-glucan. Galactomannan is another component found in some fungal cell walls.

Not all fungi have beta 1,3 D-glucan. The mnemonic BMC helps remember those that do not:

• B: Blastoises
• M: Mucor (and other Zygomycetes like Rhizopus, Absidia)
• C: Cryptococcus Testing for beta 1,3 D-glucan can be negative in infections caused by these fungi.

The galactomannan test is positive in most fungal infections, particularly associated with invasive aspergillosis. Mucor also lacks galactomannan.

The primary culture medium for fungi is Sabouraud's Dextrose Agar (SDA).

• SDA has an acidic pH of 5.6.
• It contains gentamicin to inhibit bacterial growth.
• It may contain cycloheximide to inhibit the growth of normal, saprophytic fungi, allowing pathogenic fungi to grow.
• These properties, particularly the acidic pH and addition of cycloheximide, make it useful for culturing dermatophytes.

Fungi can be visualized directly from patient samples or from culture growth using various stains.

• Gomori Methanamine Silver (GMS) stains fungi black.
• Periodic Acid Schiff (PAS) stains fungi pink.
• CalcoFluor white is a fluorescent stain that binds to chitin in the fungal cell wall. It requires a fluorescent microscope.
• Lactophenol Cotton Blue (LPCB) is commonly used for microscopy of fungi grown on culture (culture morphology). It contains lactic acid, phenol (disinfectant), cotton blue (stain), and glycerol. Glycerol acts as a hygroscopic agent, preventing the specimen from drying out.

Some Important Fungi Discussed Include

• Malassezia furfur (causes Tinea versicolor/Pityriasis versicolor). This presents as hypopigmented scaly patches.
  
  • Diagnosis is often by KOH mount of skin scrapings, which digests keratin and shows the characteristic spaghetti and meatball or banana and grapes appearance (yeast and hyphae forms).
  • It cannot be grown on regular SDA. It is lipophilic and requires olive oil for growth.
  • Colonies often appear as fried egg colonies. Malassezia furfur is one of several things that can show fried egg appearance (others include oligodendroglioma, hairy cell leukemia, seminoma/dysgerminoma, Mycoplasma).
  • Treatment includes topical/systemic azoles and sulfur ointments.
• Dermatophytes are fungi that cause infections of the skin, hair, and nails. The three main genera are Trichophyton, Microsporum, and Epidermophyton (TME).
  
  • They are identified based on the shape and presence of macroconidia and microconidia. Macroconidia shapes are pencil-shaped (Trichophyton), spindle-shaped (Microsporum), and club-shaped (Epidermophyton). Microconidia are most abundant in Trichophyton.
  • Their tissue tropism varies:
    • Trichophyton: Skin, hair, nail
    • Microsporum: Skin, hair
    • Epidermophyton: Skin, nail
  • The hair perforation test differentiates Trichophyton mentagrophytes (positive, shows wedge-shaped perforations) from Trichophyton rubrum (negative).
• Subcutaneous fungi infect tissues below the skin. The mnemonic MRCS covers four types:
  
  • Mycetoma (Mictoma)
  • Rhinosporidiosis
  • Chromoblastomycosis
  • Sporotrichosis
    • Sporotrichosis is caused by Sporothrix schenckii. It is known as rose gardener's disease, typically acquired via a thorn prick. It spreads via lymphatic channels. Microscopy shows rosettes of conidia, cigar-shaped yeast forms, and asteroid bodies (Splendore-Hoeppli phenomena - antigen-antibody reaction). Asteroid bodies are extracellular in sporotrichosis, differentiating them from the intracellular asteroid bodies seen in giant cells in sarcoidosis.
  • Rhinosporidiosis is caused by Rhinosporidium seeberi. It typically affects the nose, causing strawberry polyps. It is associated with bathing in ponds/lakes and is now reclassified as an aquatic protozoa. Diagnosis is by biopsy showing spherules or sporangia, as it is a non-cultivable organism on SDA. Treatment is surgical (polypectomy). Note: This is distinct from Rhinocladiella mackenziei, which is a fungal cause of rhino-orbital-cerebral infection, and Rhinoscleroma, a bacterial infection by Klebsiella rhinoscleromatis showing Mikulicz cells.
  • Chromoblastomycosis is associated with trauma from materials like wooden sticks. Clinically, it presents as warty, cauliflower-like lesions (verrucous dermatitis). Histopathology shows characteristic copper penny bodies (also called Medlar bodies, muriform bodies, or sclerotic bodies). Causative organisms are typically FFF sounding (Fonsecaea, Exophiala, Phialophora, Cladophialophora). Treatment is multimodality (Amphotericin B, surgery, laser).
• Dimorphic fungi exist as yeast forms at 37°C (heat) and mold forms (hyphae) at 25°C (cold). The mnemonic "Body Heat Probably Changes Shape" lists the common dimorphic fungi:
  • Body: Blastoises (Blastomyces dermatitidis). Known as North American blastomycosis (Gilchrist/Chicago disease). Shows figure of eight/broad-based budding yeast.
  • Heat: Histoplasma (Histoplasma capsulatum). Known as Ohio/Darling/Caves disease. Associated with bat and bird droppings. It is an intracellular fungus, found inside macrophages (reticuloendothelial cells). Mold form shows tuberculate macroconidia. The urine antigen test is positive.
  • Probably: Penicillium marneffei (Talaromyces marneffei). The reservoir is the bamboo rat. Produces a red pigment. Shows a broom appearance on microscopy.
  • Changes: Coccidioidomycosis (Coccidioides immitis/posadasii). Known as desert rheumatism. Associated with desert/valley history. Shows barrel-shaped arthrospores and cup-shaped spherules.
  • Shape: Sporothrix (Sporothrix schenckii). Already discussed under subcutaneous fungi.
• Systemic fungi can cause widespread disease, often in immunocompromised patients.
  • Cryptococcus neoformans/gatti cause infections like meningitis and pneumonia, especially in HIV patients. C. neoformans is associated with pigeon droppings (serotypes A, D), while C. gattii is associated with eucalyptus trees and tropical areas (serotypes B, C). Diagnosis of cryptococcal meningitis is often done using negative stain (India ink/Nigrosin) on CSF, showing unstained encapsulated yeast cells against a stained background. Mucicarmine stain is used for biopsy specimens. Bird seed/Niger agar is a selective medium for C. neoformans, producing black/brown colonies. Cryptococcus is urease positive. The Lateral Flow Assay (LFA) is a better diagnostic test than the Latex agglutination test. Treatment for cryptococcal meningitis is typically liposomal Amphotericin B plus Flucytosine, followed by fluconazole maintenance. Cryptococcus hides in the brush border in a parasitophorous vacuole ("cryptocurrency hidden"). Note: Cryptosporidium (parasite) and Cryptococcus (fungus) are different organisms causing different diseases, though both are seen in HIV patients.
  • Candida species are common causes of opportunistic infections. Candida albicans and Candida dubliniensis are particularly important.
    • They typically exist as budding yeast with pseudohyphae. C. albicans can also form true hyphae.
    • On SDA, they form creamy, pasty, yeasty colonies.
    • Different species can be differentiated on Chromagar.
    • Chlamydospores (double-walled spores) are produced on cornmeal agar by C. albicans and C. dubliniensis.
    • The germ tube test (Reynolds-Brod phenomenon) where yeast forms a tube-like outgrowth, is positive for C. albicans and C. dubliniensis.
    • C. albicans can grow in hypertonic saline and at temperatures above 45°C, while C. dubliniensis cannot.
    • In Pap smears, Candida can show a shish kebab appearance.
  • Pneumocystis jirovecii (formerly P. carinii) is a fungus causing Pneumocystis Pneumonia (PCP), especially in HIV patients with CD4 counts below 200. It is also known as plasma cell pneumonia.
    • Patients present with a dry cough.
    • Diagnosis is often via bronchoalveolar lavage (BAL), showing plasma cells pathologically and the characteristic crushed pingpong ball appearance with GMS stain microbiologically.
    • The drug of choice is Cotrimoxazole. Pentamidine is used in severe cases.

Key Parasitic Infections

Parasitology covers a diverse range of organisms, including protozoa and helminths.

• Entamoeba histolytica is an intestinal amoeba causing amoebiasis.
  
  • It causes bloody diarrhea and abdominal pain.
  • In the intestine, it forms characteristic flask-shaped ulcers.
  • It can cause amoebic liver abscesses, which contain brown pus described as having an anchovy sauce appearance.
  • Diagnosis is typically by stool examination for cysts or trophozoites. Trophozoites are seen in pus from liver abscesses.
  • The trophozoite has one nucleus with a central dot and peripheral chromatin (cartwheel appearance) and is the only parasite that eats red blood cells (erythrocytosis).
  • The cyst has 1-4 nuclei and contains chromatid bodies (made of ribonucleoprotein, stained by iron hematoxylin).
  • The infective stage is the cyst.
  • Treatment is Metronidazole plus Paromomycin (a luminal amoebicide).
  • Entamoeba coli is a non-pathogenic amoeba; its trophozoite has 1 nucleus, but its cyst has 1-8 nuclei.
• Balantidium coli is the largest intestinal protozoa and the only ciliated protozoa of the intestine.
  • It causes loose stools, fever, and abdominal pain.
  • It is found in the large intestine.
  • It exhibits rotatory motility due to cilia.
  • Both the trophozoite and cyst are binucleated, having two nuclei. The large macronucleus is kidney/bean-shaped, and there is a small micronucleus.
  • The infective stage is the cyst.
  • The treatment is Doxycycline.
• Free-living amoeba found in the environment can cause severe infections.
  • Naegleria fowleri is associated with swimming in dirty/warm fresh water. It enters the brain via the nose (cribriform plate) and causes acute, fatal Primary Amoebic Meningoencephalitis (PAM). (Nala nahana nak neural mnemonic).
  • Acanthamoeba spp. is associated with contact lens usage (causing keratitis/corneal ulcers/retinitis) and in immunocompromised individuals (causing chronic Granulomatous Amoebic Encephalitis (GAE)). The trophozoite has characteristic spikes (acanthopodia), and the cyst has a wrinkled wall. (Acantho means spike).
  • Balamuthia mandrillaris is a cousin of Acanthamoeba also causing GAE. Its trophozoite is described as fish-shaped.
  • Both Naegleria and Acanthamoeba are cultured on non-nutrient agar (NNA) with E. coli.
• Giardia lamblia (also called G. duodenalis or G. intestinalis) is an intestinal flagellate.
  • It causes foul-smelling, fatty stools (steatorrhea) and malabsorption.
  • It is found in the duodenum and is a luminal organism.
  • It exhibits falling leaf motility.
  • The infective stage is the cyst.
  • The trophozoite has two nuclei ("two eyes") and four pairs of flagella (8 total). It is described as having a tennis racket appearance.
  • The cyst has four nuclei.
  • Diagnosis can be made by stool microscopy or by sampling duodenal contents using a string test.
  • Treatment is Metronidazole or Tinidazole.
• Trichomonas vaginalis is a flagellated protozoa causing a sexually transmitted disease (STD).
  • It exists only as a trophozoite stage; there is no cyst stage. The infective stage is the trophozoite.
  • It exhibits twitching motility.
  • The trophozoite has one nucleus and five flagella (four anterior, one posterior). It can also be described as having a tennis racket appearance.
  • It is cultured on Diamond media or Lash media.
  • Clinical features include green discharge and strawberry cervix.
  • Treatment is Metronidazole, and partner treatment is essential.
• Hemoflagellates are flagellated protozoa found in the blood and tissues.
  • Trypanosoma cruzi causes South American sleeping sickness or Chagas disease ("jakas disease").
    • The vector is the kissing bug (Triatomine/Reduviid bug).
    • Clinical features include cardiomegaly (mega heart), megaesophagus, megacolon, and Romana sign (unilateral periorbital edema).
    • It can be cultured on NNN media.
    • Treatment is Benznidazole or Nifurtimox.
  • Trypanosoma brucei causes African sleeping sickness.
    • The vector is the Tsetse fly (Glossina).
    • Two types exist: West African (T.b. gambiense, chronic, causes Winterbottom sign - cervical lymphadenopathy) and East African (T.b. rhodesiense, acute, systemic).
  • Leishmania donovani causes visceral leishmaniasis (Kala-azar).
    • The vector is the sandfly (Phlebotomus).
    • Clinical features include fever of unknown origin (PUO), massive hepatosplenomegaly, and hyperpigmented skin ("kala azar").
    • Diagnosis can be made from bone marrow or spleen aspirates, showing LD bodies (Leishman-Donovan bodies), which are intracellular amastigotes within macrophages, containing a nucleus and a kinetoplast.
    • It can be cultured on NNN media.
    • A rapid diagnostic test is the RK39 antigen test.
    • Treatment is primarily liposomal Amphotericin B. Oral Miltefosine is used for post-kala-azar dermal leishmaniasis. Leishmania amastigotes, like Histoplasma, are found intracellularly in macrophages.
• Plasmodium species cause malaria, characterized by fever and chills.
  • Monocytes are typically elevated.
  • The standard stain for malaria parasites in blood smears is JSB (Jaswant Singh Bhachara) stain or Romanowsky stain (using a pH of 7.2).
  • Malaria parasites have different stages in the red blood cells, including the ring form (trophozoite) and gametocytes.
    • Plasmodium vivax: Shows one ring and one dot. The gametocyte is oval. P. vivax causes relapse due to hypnozoites (dormant forms) in the liver. The Duffy antigen on red blood cells is a receptor for P. vivax entry; its absence provides protection against P. vivax infection. Shows Schüffner's dots.
    • Plasmodium falciparum: Shows many rings and many dots. Characteristic forms include the applique/accolé form (ring on the red cell margin). The gametocyte is banana/crescent/elongated-shaped. It causes severe malaria, including algid malaria (malaria with shock/circulatory collapse). P. falciparum causes recrudescence (relapse due to inadequate treatment). Shows Maurer's clefts.
    • Plasmodium malariae: Shows a band-shaped trophozoite across the red cell. Causes recrudescence. Shows Maurer's clefts (also called Man dots).
    • Plasmodium ovale: Causes relapse. Shows James dots.
  • Gametocytes: Macro gametocytes are female (organized nucleus), and micro gametocytes are male (scattered nucleus).
  • Drug resistance is seen in India.
  • Sickle cell anemia provides some protection against malaria survival within red blood cells.
• Babesia microti is a parasite found in red blood cells.
  • It is associated with rats (reservoir) and transmitted by ticks (Ixodes).
  • Microscopy shows a Multis cross appearance (tetrad of rings) inside the red blood cells.

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