Lector Tvorko M. S. They role in human pathology. Principles of microbiological diagnosis, specific therapy.

Classification. S. aureus, S. epidermidis, S. saprophyticus

VIRULENCE FACTORS OF STAPHYLOCOCCUS AUREUS

Staphylococcus aureus Growing on Blood Agar Staphylococcus epidermidis Growing on Blood Agar Staphylococcus saprophyticus Growing on Blood Agar

Coagulase Test on Staphylococcus aureus Coagulase Test on Staphylococcus epidermidis

Staphylococcus aureus Growing on Yelk-salt Agar

Staphylococcus aureus Growing on Mannitol Salt Agar. Staphylococcus epidermidis Growing on Mannitol Salt Agar

Staphylococcus aureus Growing on DNase Agar Staphylococcus epidermidis Growing on DNase Agar

Staphylococcal Skin Infections Localized infections Folliculitis (sties, pimples, and carbuncles) result from S. aureus entering natural openings in the skin – hair follicle Impetigo of the newborn highly contagious superficial skin infection caused by S. aureus. Toxemia occurs when toxins enter the bloodstream; Scalded skin syndrome Toxic shock syndrome Figure 21.4

Staphylococcal Food Poisoning Figure 25.6 Staphylococcus aureus enterotoxin

Note gram-positive (purple) cocci in chains (arrows). Electron Micrograph of Streptococcus pyogenes

Streptococcus are gram-positive cocci classified according to their hemolytic enzymes and cell wall antigens. Group A beta-hemolytic streptococci (including Streptococcus pyogenes) are the pathogens most important to humans. Produce a number of virulence factors: M protein, erythrogenic toxin, deoxyribonuclease, streptokinases, hyaluronidase. Figure 21.5

Streptococcus in chains (Gram stain)

hemolysis reaction - sheep blood agar (alpha) (alpha) partial hemolysis partial hemolysis  green color (beta) (beta) complete clearing complete clearing A and B A and B (gamma) (gamma) no lysis no lysis White colonies

Beta Hemolysis on Blood Agar (Indirect Lighting)

Alpha Hemolysis on Blood Agar (Indirect Lighting)

Gamma Reaction on Blood Agar A Plate of Blood Agar showing Alpha, Beta, and Gamma Hemolysis (Indirect Lighting)

Streptococcal pharyngitis Strep throat Streptococcus pyogenes  Resistant to phagocytosis  Streptokinases lyse clots  Streptolysins are cytotoxic Pharyngitis - Scarlet Fever Erythrogenic toxin produced by lysogenized S. pyogenes Diagnosis by indirect agglutination Figure 24.3

Erysipelas infects the dermal layer reddish patches Can progress to local tissue destruction Enter the bloodstream Impetigo isolated pustules Streptococcal toxic shock syndrome Cellulitis, myositis and necrotizing fasciitis Streptococcal Skin Infections Figure 21.6, 7

The inner layer of the heart is the endocardium. Inflammation of the endocardium Endocarditis Subacute bacterial endocarditis – from microbs in the mouth.( Arises from a focus of infection, such as a tooth extraction).  alpha-hemolytic streptococci  staphylococci  enterococci Preexisting heart abnormalities are predisposing factors. Signs include fever, anemia, and heart murmur. Acute bacterial endocarditis Staphylococcus aureus The bacteria cause rapid destruction of heart valves Bacterial Infections of the Heart

Rheumatic fever is an autoimmune complication of streptococcal infections. Rheumatic fever is expressed as arthritis or inflammation of the heart. It can result in permanent heart damage. Rheumatic fever can follow a streptococcal infection, such as streptococcal sore throat. Streptococci might not be present at the time of rheumatic fever. Prompt treatment of streptococcal infections can reduce the incidence of rheumatic fever. Penicillin is administered as a preventive measure against subsequent streptococcal infections. Antibodies against group A beta-hemolytic streptococci react with streptococcal antigens deposited in joints or heart valves or cross-react with the heart muscle. Rheumatic Fever Figure 23.5

Dental Caries Streptococcus mutans, found in the mouth uses sucrose form dextran from glucose lactic acid from fructose. Bacteria adhere to teeth and produce sticky dextran, forming dental plaque. Acid produced during carbohydrate fermentation destroys tooth enamel at the site of the plaque. Gram-positive rods and filamentous bacteria can penetrate into dentin and pulp. Caries are prevented by restricting the ingestion of sucrose and by the physical removal of plaque. Figure 25.4

Pneumomoccal Pneumonia Streptococcus pneumoniae: Gram-positive encapsulated diplococci Diagnosis by culturing bacteria Penicillin is drug of choice Figure 24.13

Scanning Electron Micrograph of Streptococcus pneumoniae

Streptococcus pneumoniae (diplococcus). Fluorescent stain

Streptococcus pneumoniae on Blood Agar (Indirect Lighting)

A bile esculin slant before inoculation. Enterococcus faecalis growing on a bile esculin slant. Note black color. Enterococcus faecalis in a Blood Culture

Meningococci

Meningococci Gram staining

Identification of Neisseria meningitidis : Carbohydrate Utilization Colonies of Neisseria meningitidis on Blood agar

Clinical symptoms of meningococcal infection

Pathological changes of brain (meningococcal infection)

Spinal fluid

Diagnosis and Treatment of the Most Common Types of Bacterial Meningitis Diagnosis is based on Gram stain and serological tests of the bacteria in CSF. Cultures are usually made on blood agar and incubated in an atmosphere containing reduced oxygen levels. Cephalosporins may be administered before identification of the pathogen.

Neisseria gonorrhoeae Gram -, diplococcus Gram stain pus, intracellular diplococcus Virulence factors Pili Initial attachment Antigenic and phase variation Opacity protein (Opa) Tighter contact and invasion Antigenic variation LOS (lipooligosaccharide, lack O- Ag) Inflammatory, major cause of symptom IgA protease

Affected Populations STI Rates World-Wide

Gonococci (methylen blue staining)

Gonococci

Electron Micrograph of Neisseria gonorrhoeae

Colonies of gonococci onto blood agar

Identification of Neisseria : MTM Chocolate Agar with a Taxo (Oxidase) Disc

Identification of Neisseria gonorrhoeae : Carbohydrate Utilization A Positive Direct Fluorescent Antibody Test for Neisseria gonorrhoeae

Neisseria gonorrhoeae (3) Pili: facilitate attachment, impede phagocytosis Lipopolysaccharide: marked endotoxin activity; local cytopathic effect IgA protease: cleaves IgA-1 subclass of immunoglobulins

Neisseria gonorrhoeae (4) Porin (“Por”, formerly protein I): may facilitate endocytosis Opacity proteins (“Opa,” formerly protein II): contribute to attachment to human cells Reduction-modifiable protein (“Rmp,” formerly protein III: stimulates blocking antibodies that reduce serum bactericidal activity

GC Sexual Transmission Efficiently transmitted by sexual contact Greater efficiency of transmission from male to female  Male to female: %  Female to male: % Vaginal & anal intercourse more efficient than oral Can be acquired from asymptomatic partner Increases transmission and susceptibility to HIV 2-5 fold

Gonorrhea Many asymptomatic Reason for spread Male- Urethritis, urethral discharge Female - Endocervicitis, discharge, dysuria, bleeding Pharyngitis Proctitis Disseminated gonococcal infection (DGI) Pustular skin lesions Septic arthritis Pelvic inflammatory disease (PID) Endometritis, salpingitis, peritinitis Infertility, ectopic pregnancy Ophthalmia neonatorum

Gonococcal Infections in Women Cervicitis Urethritis Proctitis Accessory gland infection (Skene, Bartholin) Pelvic inflammatory disease (PID) Peri-hepatitis (Fitz-Hugh-Curtis) Pregnancy morbidity Conjunctivitis Many infections asymptomatic n Pharyngitis n DGI

Gonococcal Cervicitis Incubation 3-10 days Symptoms: u Vaginal discharge u Dysuria u Vaginal bleeding Cervical signs : u Erythema u Friability u Purulent exudate

Pelvic Inflammatory Disease STD Atlas, 1997 Sx: lower abdominal pain Signs: CMT, uterine/ adnexal tenderness, +/- fever Laparoscopy may show hydrosalpinx, inflammation, abscess, adhesions Adhesions Tube

Gonococcal Infections in Men Urethritis Epididymitis Proctitis Conjunctivitis Abscess of Cowper’s/Tyson’s glands Seminal vesiculitis Prostatitis Many infections asymptomatic Pharyngitis DGI Urethral stricture Penile edema

Gonococcal Urethritis Incubation 2-7 days Abrupt onset of severe dysuria Purulent urethral discharge Most urethral infections symptomatic

Epididymitis  Swollen painful epididymis  Urethritis  Epididymal tenderness or mass on exam Epididymitis

Gonococcal Infections in Women & Men Urethritis Proctitis Pharyngeal infections Conjunctivitis Disseminated Gonococcal Infection

Blenorrhea

Gonorrhea Diagnosis Intracellular Gram negative diplococci in discharge Growth on selective media, oxidase positive colonies Flourescent antibody Treatment Cover for probable association with C. trachomatis

GC Diagnostic Methods Gram stain smear Culture Antigen Detection Tests: EIA & DFA Nucleic Acid Detection Tests Probe Hybridization Nucleic Acid Amplification Tests (NAATs) Hybrid Capture

Gonorrhea Diagnostic Tests Gram stain (male urethra exudate) DNA probe Culture NAATs * Sensitivity 90-95% 85-90% 80-95% 90-95% Specificity  95%  99%  98% * Able to use URINE specimens

Gram Stain for GC: Urethral Smear Numerous PMNs Gram negative intracellular diplococci

Gram Stain for GC: Cervical Smear PMN with Gram negative intracellular diplococci

GC Culture Requires selective media with antibiotics to inhibit competing bacteria (Modified Thayer Martin Media, NYC Medium) Sensitive to oxygen and cold temperature Requires prompt placement in high-CO2 environment (candle jar, bag and pill, CO2 incubator) In cases of suspected sexual abuse, culture is the only test accepted for legal purposes

GC Culture Candle Jar

GC Culture Specimen Streaking Cervical and Urethral

GC Culture After 24 Hours

Treatment

Prevention Abstinence Safe/”smart” sex Barrier contraceptives Educational programs Reduce misuse of antimicrobials