Comprehensive Specialty Microbiology Study Guide for Krok-1

Foreword and General Educational Context

The provided informational materials are tailored for students of medical and dental faculties at medical universities with III-IV levels of accreditation. These notes are aligned with the official Program of Special Microbiology and are designed to facilitate preparation for the licensing exam known as Krok-1. Due to the vast volume of scientific information in modern microbiology, it is vital to systematize knowledge regarding the classification, morphology, and ultrastructure of bacteria. Specific attention is given to the reproductive features and cultivation methods of Rickettsiae, Chlamydiae, and Mycoplasmas. The content emphasizes infectious disease prevention, therapy, and modern laboratory diagnostics as used in the Testing Center of the MoH of Ukraine.

Bacteriology of Staphylococci

Staphylococci are chemoorganotrophs characterized by both oxidative and fermentative metabolism. Morphologically, they appear as Gram-positive cocci, approximately $0.5 - 1.5\, \mu m$ in diameter, typically arranged in irregular clusters resembling bunches of grapes. They are non-motile, non-spore-forming, and may possess a delicate capsule within the host organism. Their cell walls contain peptidoglycan and teichoic acids. On a dense nutrient medium, they form round, convex, pigmented colonies (golden, lemon-yellow, or white) with smooth edges. In liquid media, they cause uniform turbidity. The pigment is lipochromic and requires oxygen and specific media components (blood, carbohydrates, or milk) for expression. They are facultative anaerobes and are relatively undemanding regarding nutrient media, though they are halophilic and can grow in high concentrations of salt (7-10% $NaCl$), making salt-based media like salt agar or egg-yolk-salt agar (Chistovich's medium) selective for this genus.

Staphylococcus aureus (golden staphylococcus) is a primary pathogen in this group. For epidemiological purposes, such as tracing the source of an intrahospital infection outbreak, phagotyping is the definitive method. A phagotype is a set of bacterial strains characterized by identical sensitivity to a specific set of bacteriophages. This is more effective than antibiograms or biochemical testing for identifying a single source among personnel or items. S. aureus is distinguished from other species like S. epidermidis by its ability to synthesize plasmocoagulase (the coagulase test), ferment mannitol under anaerobic conditions, and express lecithinase activity (forming a yellow opaque zone on egg-yolk-salt agar). Lecithinase activity is detected on Chistovich's medium because the egg-yolk emulsion becomes transparent, but the presence of lipase/lecithinase creates an opaque zone around the colony. Etiological significance in food poisoning is often proven by the detection of enterotoxins.

Bacteriology of Streptococci and Pneumococci

Streptococci are Gram-positive cocci, $0.6 - 1.0\, \mu m$ in size, arranged in chains of varying lengths. They are facultative anaerobes and are more fastidious than staphylococci, requiring media enriched with glucose, serum, or blood. In sugar broth, they typically form a bottom sediment while the rest of the broth remains clear. They are classified by their hemolytic activity on blood agar: $\beta$-hemolytic (complete hemolysis zone), $\alpha$-hemolytic (incomplete green zone), and $\gamma$-hemolytic (no hemolysis). Streptococcus pyogenes is associated with scarlet fever, which presents with high fever, sore throat, an enlarged "crimson tongue," and a diffuse red rash (macular) that spares the nasolabial triangle. The Dick reaction is used for diagnosis: an intradermal injection of erythrogenic toxin causes a localized red reaction in susceptible individuals, while a "negative" result (no reaction) occurs in those with immunity (antitoxic antibodies). Acute post-streptococcal glomerulonephritis is a common complication, occurring as a Type III immune complex allergic reaction affecting the basement membrane of renal corpuscles.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive diplococcus. The cells are characteristically lanceolate or oval, arranged in pairs with the tapered ends facing outward. Inside the host, they are surrounded by a thick polysaccharide capsule that resists phagocytosis. In pure culture, they may form short chains. They are catalase- and oxidase-negative and grow better in an atmosphere with 5-7% $CO_2$. In dental microbiology, Streptococcus mutans and lactobacilli are primary associations involved in cariogenesis and enamel demineralization (manifesting as "white spots"). Dental infections are often treated with structure-containing $\beta$-lactam rings, such as Benzylpenicillin.

Neisseria: Meningococci and Gonococci

Neisseria gonorrhoeae (gonococcus) is the causative agent of gonorrhea and ophthalmia (blennorrhea) in newborns. Morphologically, they are Gram-negative, bean-shaped diplococci found both intracellularly (within neutrophils) and extracellularly. They ferment only glucose to acid. Diagnosis of acute cases relies on microscopy, while chronic cases often require the Complement Fixation Test (CFT), also known as the Bordet-Gengou reaction. Reinfection is possible as immunity is not long-lasting. Ciprofloxacin (a fluoroquinolone) is a primary treatment choice. In newborns, blennorrhea is an acute purulent inflammation of the eye's mucous membrane acquired during childbirth.

Neisseria meningitidis (meningococcus) causes nasopharyngitis, meningitis, and meningococcemia. Like gonococci, they are Gram-negative, coffee-bean-shaped diplococci. Meningococcemia is a severe form characterized by sudden high fever, hemorrhagic skin rash, oliguria, and adrenal insufficiency (Waterhouse-Friderichsen syndrome), which can be fatal. For laboratory identification, nasopharyngeal mucus, blood, or cerebrospinal fluid (CSF) is collected. They are highly sensitive to environmental factors and require specialized media (serum agar) and careful transport to ensure survival.

Enterobacteriaceae: Escherichia, Salmonella, and Shigella

Escherichia coli is used as a sanitary indicator of fecal contamination. Enteropathogenic E. coli (EPEC), such as strains with the antigenic structure O55:K59 or O111, cause coli-enteritis in infants. On Endo agar, lactose-fermenting E. coli form bright red colonies with a metallic luster. This color change is due to the release of formic acid during lactose fermentation, which reacts with the Schiff reagent (basic fuchsin decolorized by sodium sulfite) to restore the red color of the fuchsin.

Salmonella species cause typhoid fever, paratyphoid, and food poisoning. Typhoid fever is characterized by bacteremia during the first week; thus, blood culture is the diagnostic priority. The Widal test is a classic agglutination reaction used for serodiagnosis by detecting antibodies against O and H antigens. The detection of Vi-antibodies using the Passive Hemagglutination Assay (PHA) is critical for identifying asymptomatic carriers. If a patient’s condition worsens following Levomycetin (chloramphenicol) treatment (fever spikes to $39.6^o C$), it is likely due to the massive release of endotoxins from the destroyed bacteria.

Shigella species are the agents of bacterial dysentery. Major species include S. dysenteriae, S. flexneri, and S. sonne. Clinical presentation involves colitis with bloody, mucoid stool and tenesmus. Diagnosis is primarily via the bacteriological method (isolating pure culture from feces). Specific prophylaxis for contacts in outbreaks (especially S. sonne) may involve the use of dysentery bacteriophage. Retrospective diagnosis is performed using PHA to determine antibody titers in the serum.

Vibrio Cholerae and Borrelia

Vibrio cholerae is the agent of cholera, presenting with "rice-water" stool, severe dehydration, and anuria. The bacteria are Gram-negative, comma-shaped, and possess a single polar flagellum (monotrichous). They are highly motile, which is observed using the "hanging drop" or "crushed drop" technique via phase-contrast or dark-field microscopy. Culturally, they are halophilic and alkaliphilic, growing rapidly (within 6 hours) on 1% alkaline peptone water to form a delicate bluish film on the surface. They also grow on alkaline agar.

Borrelia and Leptospira are spiral-shaped bacteria. Borrelia recurrentis causes relapsing fever, characterized by alternating periods of high fever and apyrexia. It is transmitted by lice. Laboratory diagnosis involves staining a "thick drop" of blood using the Romanovsky-Giemsa method, revealing blue-violet spirals with pointed ends. Leptospira interrogans are thin spirals resembling the letters 'C' or 'S' and are also typically visualized via dark-field microscopy.

Corynebacterium Diphtheriae

Corynebacterium diphtheriae causes diphtheria, a disease characterized by the formation of thick, grayish fibrous films (pseudomembranes) on the tonsils or respiratory tract. These films are difficult to remove and leave bleeding tissue injuries. Morphologically, they are Gram-positive rods with club-shaped ends due to the presence of volutin granules (Babes-Ernst bodies). Staining methods include Neisser (yellow rods with dark-blue poles) and Loeffler (blue rods). For toxigenicity testing, the gel precipitation reaction (Elek's test) is used; lines of precipitation form where the exotoxin meets antitoxic serum. Toxigenicity is acquired through phage conversion (lysogeny), where a temperate bacteriophage carries the tox-gene. Treatment involves the immediate administration of antitoxic anti-diphtheria serum. Prophylaxis is achieved via DTP vaccine (containing diphtheria toxoid).

Mycobacterium Tuberculosis and Leprae

Mycobacterium tuberculosis is an acid-fast bacterium due to the high lipid content in its cell wall. It is stained using the Ziehl-Neelsen method (appearing bright red against a blue background). Cultivation on selective media like Lowenstein-Jensen takes 3-4 weeks (slow growth). To increase the detection probability in sputum, enrichment methods like homogenization and flotation (using chemicals like caustic soda and xylol) are used. The Mantoux test is a diagnostic tool using tuberculin (PPD); a positive skin reaction (papule $>5\, mm$) indicates delayed-type hypersensitivity (Type IV). A negative Mantoux test in a child suggests the absence of immunity and indicates the need for BCG vaccination (live attenuated vaccine). Anti-tuberculosis chemotherapy involves Isoniazid (inhibits mycolic acid synthesis; side effects include peripheral neuritis), Rifampicin (inhibits RNA polymerase; causes orange-red discoloration of urine/tears), and Streptomycin (aminoglycoside; can cause ototoxicity).

Mycobacterium leprae causes leprosy. It is also acid-fast and typically found in biopsies of skin or mucous membrane lesions, arranged in clusters resembling "packs of cigars."

Pathogenic Anaerobes: Botulism, Tetanus, and Gas Gangrene

Anaerobic bacteria require oxygen-free conditions and are often cultivated on Kitt-Tarozzi medium.

Clostridium botulinum: The agent of botulism, often associated with contaminated homemade canned goods or dried fish. Morphologically, they resemble a "tennis racket" due to subterminal spores. Pathogenicity is driven by a potent exotoxin that causes neurological symptoms: diplopia (double vision), dysphagia (difficulty swallowing), and respiratory failure. Diagnosis includes a neutralization reaction in white mice to identify the toxin type. Treatment requires polyvalent antitoxic serum.

Clostridium tetani: The agent of tetanus. It is a Gram-positive rod with a terminal spore, giving it a "drumstick" appearance. It is found in soil and enters through contaminated wounds. Clinical signs involve trismus (lockjaw), risus sardonicus (sardonic grin), and opisthotonus (muscle spasms). Prophylaxis for injuries includes tetanus toxoid (active) and anti-tetanus serum or immunoglobulin (passive).

Clostridium perfringens: One of the agents of gas gangrene (anaerobic wound infection). These are Gram-positive, spore-forming anaerobic bacilli. Culturally, on blood-glucose agar, they form colonies surrounded by a zone of hemolysis. They lack catalase and peroxidase, making them sensitive to hydrogen peroxide.

Zoonotic and Especially Dangerous Infections

Yersinia pestis: The causative agent of the plague. It is a Gram-negative, ovoid rod with bipolar staining and a delicate capsule. It is transmitted by flea bites from rodent reservoirs. Colonies on agar look like "lace handkerchiefs" with a dense center and a jagged border. The vaccine of choice is the live EV vaccine.

Brucella: Agents of brucellosis (B. melitensis, B. abortus, B. suis). They are small, Gram-negative, capnophilic (require $CO_2$) coccobacilli. Transmission often occurs via unpasteurized milk. The Burnet skin test (using brucellin) and the Wright agglutination test are used for diagnosis.

Bacillus anthracis: The agent of anthrax. It is a large, non-motile Gram-positive streptobacillus with cut-off ends resembling "bamboo stalks." Within the host, it forms a capsule (Burri-Gins stain); in the environment, it forms highly resilient spores that can survive in soil for over 50 years. The Ascoli thermoprecipitation test is used to detect anthrax antigens in animal raw materials (e.g., hides). The STI live vaccine is used for prevention.

Pathogenic Protozoa and Fungi

Entamoeba histolytica: The agent of amoebic dysentery. Vegetative forms contain phagocytosed erythrocytes (erythrophage). Cysts have 4 nuclei. It causes "raspberry jelly" stools. Treatment: Metronidazole.

Giardia lamblia: Found in the duodenum. These are pear-shaped organisms with 2 nuclei, 4 pairs of flagella, and a suction disc. They cause duodenitis and enteritis.

Trichomonas vaginalis: A pear-shaped flagellate with an undulating membrane and a posterior spike. It causes urogenital trichomoniasis. Trichomonas tenax is found in the oral cavity in gingivitis.

Toxoplasma gondii: A crescent-shaped intracellular parasite. It can cause congenital defects and intrauterine death. Diagnosis involves serological tests like PHA or the Sabin-Feldman dye test.

Leishmania: L. donovani causes visceral leishmaniasis (enlarged liver/spleen), and L. tropica causes cutaneous leishmaniasis (ulcers). Transmitted by sandflies (mosquitos).

Plasmodium: The agent of malaria, transmitted by the Anopheles mosquito. Diagnosis is by microscopy of blood smears (thick and thin films). Quinine or Chingamin (Chloroquine) is used for treatment and prophylaxis.

Candida albicans: A yeast-like fungus that causes candidiasis (thrush). It forms pseudomycelium and budding oval cells. It is a common complication of long-term antibiotic therapy. Cultivated on Sabouraud agar. Treatment: Fluconazole, Nystatin, or Amphotericin B.

Sanitary Microbiology and Indices

Sanitary bacteriological analysis of water involves several key metrics defined by state standards. The Total Microbial Count (TMC) should not exceed 100 bacteria per $1.0\, ml$ of tap water. The Coli-index is the number of bacteria of the E. coli group (coliforms) in $1\, Liter$ of water; For tap water, it should be no more than 3. The Coli-titer is the smallest volume of water (in $ml$) containing one E. coli; it should be at least $300\, ml$.

Calculations example:

• If 2 red colonies grow from $500\, ml$ of water on Endo agar:
• $Coli\text{-}index = \frac{2 \times 1000}{500} = 4$
• $Coli\text{-}titer = \frac{500}{2} = 250$

In the air of enclosed spaces, the presence of hemolytic staphylococci or streptococci serves as a direct indicator of epidemiological danger.