BACTE 2ND LECTURE.pdf

CLINICAL BACTERIOLOGY – 2ND LECTURE EXAM TERMINOLOGIES: ANTIMICROBIAL SUSCEPTIBILITY TESTING MINIMUM INHIBITORY CONCENTRATION (MIC): lowest concentration...

CLINICAL BACTERIOLOGY – 2ND LECTURE EXAM TERMINOLOGIES: ANTIMICROBIAL SUSCEPTIBILITY TESTING MINIMUM INHIBITORY CONCENTRATION (MIC): lowest concentration of the antimicrobial agent which inhibits bacterial growth. It is performed on bacteria isolated from clinical specimens to determine which antimicrobial agents might be effective in treating infections caused MINIMUM LETHAL CONCENTRATION (MLC): lowest concentration of by the bacteria. the antimicrobial agent which kills the bacterial growth when subculture to a fresh medium Susceptibility testing is usually performed by disk diffusion or dilution (minimum inhibitory concentration method). MINIMUM BACTERICIDAL CONCENTRATION (MBC): lowest concentration of the antimicrobial agent needed to kill the bacterial growth. BROTH DILUTION METHOD BREAKPOINT (CUTOFF): refers to the concentration of an antimicrobial It involves challenging the organism of interest with antimicrobial agents agent that coincides with a susceptible or intermediate minimum inhibitory in a broth environment (Mueller-Hinton broth). concentration (MIC) breakpoint for a particular drug. A specific amount of antibiotic is prepared in a decreasing concentration TRAILING GROWTH: involves heavy bacterial growth at lower in broth by serial dilution technique, and standard amount of the test concentrations followed by one or more wells that show greatly reduced organism is inoculated. growth in the form of a small button or a light haze. Absence of turbidity of broth signifies inhibition of bacterial growth by the SKIPPED WELLS: involves growth at higher concentrations and no antibiotics being tested. growth at one or more of the lower concentrations; it may indicate PRINCIPLE: to determine the lowest concentration of the antimicrobial contamination, improper incubation, improper concentration of the drug (MIC) required to inhibit bacterial growth. antimicrobials and presence of unusual resistant isolate. PREFERRED METHOD: serial two-fold dilution concentration (µg/mL) AGAR DILUTION METHOD STANDARD INOCULUM SIZE: 5X105 CFU/mL Can be used to determine MIC and MLC concentrations It is the reference method for testing anaerobes and N. gonorrheae MH broth with 2% NaCl- to improve detection of oxacillin-resistant because it tends to lyse in broth media, resulting in false-susceptible. staphylococci Used for research studies only because plate preparation is laborious. MH broth with 2% to 5% lysed horse blood for streptococci MEDIA FOR ANAEROBES: Brucella-laked SBA blood (Wadsworth method) incubated at 35ºC for 48 hours. SHELF LIFE OF AGAR DILUTION PLATE: one week (for most antimicrobial agents) at 2-8 ºC. SUSCEPTIBILITY MEDIUM: MHA (aerobes); MHA = 5% SB (fastidious bacteria) STANDARD INOCULUM: 1x104 CFU/mL PROCEDURE: o A standard inoculum of bacteria is spot-inoculated onto a 100-mm plate. o A series of plates containing varying concentrations of each antimicrobial and control plates are prepared o One or more bacterial isolates (up to 32 isolates) are tested per plate; MIC can also be determined DISK DIFFUSION METHOD (KIRBY-BAUER METHOD TEST) It is limited to aerobic and facultatively anaerobic bacteria Quantitative method which provides the greatest flexibility and cost- effectiveness – it allows the laboratory to test any 12 antimicrobial agents on a 150-mm MHA plate. It depends on the formation of a gradient of antimicrobial concentrations as the antimicrobial diffuses into the agar – the drug concentration decreases at increasing distances from the disk. PRINCIPLE: the larger zone of inhibition, the lower the MIC – the zone of inhibition is inversely related to MIC BROTH MACRODILUTION SUSCEPTIBILITY STANDARD MEDIUM: Mueller Hinton Agar (MHA) SUSCEPTIBILITY MEDIUM: MH broth (non-fastidious bacteria) STANDARD INOCULUM SIZE: 1.5 X 108 CFU/mL – McFarland STANDARD INOCULUM SIZE: 5X105 CFU/mL PROCEDURE: filter paper disks impregnated with various antimicrobial INCUBATION TIME: 16-24 hours (overnight) at 35ºC agents of specific concentrations are carefully placed on an agar plate ADVANTAGE: To test microbials not included in the routine test or for previously inoculated with bacteria being tested. fastidious bacteria; use when MBC endpoints need to be subsequently determined. DISADVANTAGE: Impractical to use if there are several antimicrobial agents or isolates to be tested. TURBIDITY STANDARD 0.5% Mc Farland Turbidity Standard (Barlum Sulfate Suspension) o 99.5 mL of 1% H2SO4 + 0.5 mL of 1.175% barium chloride o Equivalent to 1.5% x 108 colony forming units (CFU)/mL  o Pure cultures are grown or are directly prepared from agar plates to STAPHYLOCOCCI match the turbidity of the 0.5% Mc Farland standard. The genus name is derived from the Greek word staple meaning READING OF PLATES AND INTERRETATION OF RESULTS “bunches of grapes” Gram-positive cocci that belong to the family Micrococcaceae 1. The lawn of growth must be confluent or almost confluent. Normal habitat is the skin, mucous membrane, and intestine 2. Provided growth is satisfactory, the diameter of each inhibition zone is Catalase-producing bacteria: facultatively anaerobic (except S. measured using a [calibrated] caliper or ruler. saccharolyticus – obligate anaerobe) 3. Plates are examined 2-3 inches above a black, nonreflecting surface Non-motile, non-spore forming ang glucose fermenter and the zones are measured from the back side of the plate. Significant human species: Staphylococcus aureus, Staphylococcus 4. Transmitted light rather than reflecting light will improve the accuracy epidermidis, Staphylococcus saprophyticus, Staphylococcus hominis, of tests with penicillinase-resistant-penicillin Staphylococcus hemolyticus 5. For media containing blood, the plate is examined with the lid MICROSCOPY: spherical cells that appear singly, in pairs, and in removed. clusters 6. The appearance of individual colonies is unacceptable. CULTURE: colonies (4-8 mm) on agar plate appear creamy, white, or 7. The zone size of a motile, swarming organism such as Proteus light gold and “buttery looking;” other species have gray colonies; some should be ignored (thin film of growth). species are β-hemolytic (S. aureus) 8. Discontinuous poor growth or tiny colonies near the end of the zone size are also ignored. DIFFERENTIAL TESTS BETWEEN STAPHYLOCOCCI AND MICROCOCCI 9. Colonies with a clear zone should not be ignored – these colonies may occur as a result of contamination or testing a mixed culture. The BACITRACIN / TAXO A DISK TEST – 0.04 units bacitracin; performed original colony should be retested. on BAP and MHA. 10. Zone measurement is compared with the interpretative tables of o Result: CLSI, and results are interpreted as susceptible, intermediate, ▪ Micrococci = susceptible (>10 mm zone of inhibition) resistant, or nonsusceptible. ▪ Staphylococci = resistant 11. Quality control plates should be read prior to reading results of a FURAZOLIDONE SUSCEPTIBILITY TEST – 100 µg furazolidone; patient isolates to determine whether the test was performed performed on BAP. correctly. o Result: ▪ Staphylococci = susceptible (>15 mm zone of inhibition) SUSCEPTIBLE, INTERMEDIATE, RESISTANT ▪ Micrococcus = resistant (6-9 mm) SUSCEPTIBLE: patient’s infecting organism should respond to therapy MODIFIED OXIDASE / MICRODASE TEST with that antimicrobial agent using the recommended dosage for the site of o Reagent paper: tetramethyl-p-phenylenediamine in infection; indicated by the presence of zone of inhibition around the dimethylsulfoxide antibiotic disk o (+) result: blue color within 2 minutes = Micrococci INTERMEDIATE: the microorganism falls into a range of susceptibility in LYSOSTAPHIN SENSITIVITY TEST which the MIC approaches or exceeds the level of antimicrobial agent that o Result: 10-16 mm zone of inhibition = Staphylococci (sensitive), can be achieved and for which clinical response is likely to be less than Micrococci (resistant) with a susceptible strain. o Note: Staphylococcus aureus is lysed with lysostaphin RESISTANT: the microorganism is inhibited by the usually achievable GLUCOSE UTILIZATION concentrations of the antimicrobial agent based on the dosages normally o PRINCIPLE: if CHO is fermented → ACID used with that drug. o MEDIUM: Hugh & Leiffson Medium o pH INDICATOR: Bromthymol blue CAUSES OF FALSE-RESISTANCE: o ALKALINE: Blue o ACID: Yellow Use of unduly heavy inoculum of cultures or undiluted specimen materials. TEST TO DIFFERENTIATE STAPHYLOCOCCUS AND MICROCOCCUS TEST STAPHYLOCOCCUS MICROCOCCUS Late examination of test plates after zones have become overgrown Aerobic growth Facultative anaerobe Obligate anaerobe Use of disc with inadequate drug concentration due to prolonged storage; Lysostaphin Sensitive Resistant failure to refrigerate as from disc container opened frequently Bacitracin Resistant Sensitive Use of wrong organism Modified Oxidase Negative Positive Glucose Utilization Fermenter Oxidizer FACTORS AFFECTING ZONE OF INHIBITION: STAPHYLOCOCCUS AUREUS The amount of inoculum or test organism Thickness of the susceptibility agar plate (4 mm) Most virulent species; coagulase (+) The growth rate of the test organism Aureus means “gold” The pH of the medium (7.2-7.4) Culture: golden yellow pigment (lipochrome); β-hemolytic on BAP The number of disk per plate (12 disk / 150 mm plate) It can be cultivated with added 7.5 – 10% NaCl (halophilic organism) The concentration of divalent cations Chiefly responsible for the various skin, wound, and deep tissue infections E-TEST ASSOCIATED DISEASES AND INFECTIONS It is a dilution test based on the diffusion of a continuous concentration gradient of an antimicrobial agent from a plastic strip into an agar medium TOXIC INDUCED DISEASES: food poisoning, scalded skin syndrome It uses thin plastic strips that are impregnated on to the under surface (SSS), and toxic shock syndrome (TSS) with an antimicrobial concentration gradient and are marked on the upper o SSS: it is an extensive exfoliative dermatitis that occurs primarily in surface with a continuous MIC concentration index or scale. newborns and previously healthy individuals o TSS (Enterotoxin F): rare but potentially fatal, multisystem disease (+) result: ellipse of growth of inhibition characterized by a sudden onset of fever, chills, vomiting, diarrhea, MINIMUM BACTERICIDAL CONCENTRATION TEST muscle aches, and rash, which can quickly progress to hypotension and shock. (associated with the use of tampons) It is an in-vitro determination of the amount of antimicrobial agent required BACTEREMIA / SEPSIS to kills as well as inhibit the bacteria URINARY TRACT INFECTION (UTI) It is performed together with a broth microdilution or microdilution MIC ACUTE BACTERIAL ENDOCARDITIS test – a 0.01 mL aliquot of each clear MIC tube or well is subcultured to an CUTANEOUS INFECTIOUS: folliculitis, furuncles (boils), carbuncles, agar medium impetigo and purulent abscess. STANDARD INOCULUM SIZE: 5x105 CFU/mL o Folliculitis: mild inflammation of hair follicle or sebaceous gland FINAL DILUTION: 1:1000 (0.01 mL) o Furuncles: large, raise, superficial abscesses; can be an extension DILUTION FACTOR: 1:10, 000 (104) of folliculitis o Carbuncles: develop from multiple furuncles which may progress GRAM-POSITIVE COCCI STAPHYLOCOCCI to deeper tissues; patients may have fever and chills (systemic infection)  o Impetigo: superficial cutaneous infection characterized by crusty ▪ Any negative slide test should be confirmed with the tube lesions and vesicles surrounded by a red border; common to children method o Osteomyelitis: secondary to bacteremia ▪ (+) result: clot / coagulum formation within 30 seconds o Septic arthritis (children) ▪ Other coagulase (+) organisms: S. lugdunensis and S. schleiferi ENZYMES AND TOXINS PRODUCED (VIRULENCE FACTORS) TUBE METHOD: considered a sensitive method; definitive test o It detects extracellular / unbound / free coagulase CATALASE: a heme enzyme, that catalyzes the decomposition of H2O2 o Procedure: inoculate a tube containing plasma and incubate at to water and oxygen 35ºC o Aerobic catalase test: using 3% H2O2 o (+) result: clot / coagulum formation after 1-4 hours of incubation o Anaerobic catalase test: using 15% H2O2 o if no clot appears after 4 hours of incubation, the tube should be left o Catalase test: used to differentiate staphylococci (+) from at room temperature for an additional 20 hours of incubation streptococci (-) o Other tube coagulase (+) organisms: S. hyicus, S. intermedius, o Pseudocatalase reaction: Aerococcus, Enterococcus, Rothia S. delphini, S. schleiferi subsp., Coagulans (formerly Stomatococcus). MANNITOL FERMENTATION TEST: used to differentiate pathogenic COAGULASE: it coagulates the fibrinogen in plasma, promotes the from nonpathogenic staphylococci formation of fibrin layer around the staphylococcal abscess thereby o MSA (1% Mannitol + 7.5% NaCl) protecting the bacteria from phagocytosis o pH indicator: Phenol red o It reacts with a thermostable thrombin-like molecule called o Result: (+) yellow color – Staphylococcus aureus (colonies are coagulase-reacting factor (CRF) to form coagulase-CRF complex. surrounded by yellow halo) o 2 TYPES o Staphylococcus saprophyticus – some strains also ferment ▪ Cell-bound coagulase / clumping factor: it is bound to a cell mannitol (resembles S. aureus on MSA) wall; it causes bacterial cells to agglutinate in plasma; it clots TELLURITE GLYCINE AGAR: (+) jet black colonies of S. aureus human, rabbit, or pig plasma. POLYMYXIN SENSITIVITY TEST: (+) result: Resistant – S. aureus ▪ Unbound coagulase / free coagulase: it is an extracellular VOGES – PROSKAUER TEST: It differentiates S. aureus (+) from S. enzyme; free from the cell wall. It causes clot formation when intermedius (-) bacterial cells are incubated with plasma. o (+) result: acetoin / acetylmethyl carbinol production – pink color HYALURODINASE (SPREADING-FACTOR ENZYME): it enhances the DNAse TEST: used to detect DNAse activity of S. aureus. invasion and survival in tissue; breaks down hyaluronic acid present in the o Medium and reagent: DNA medium and methyl green dye intracellular ground substance of connective tissues, resulting to spread to o (+) result: clearing of the dye (clear zone) bacteria METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) STAPHYLOKINASE (FIBRINOLYSIN): it has fibrinolytic activity; o It is acquired after prolonged hospital stay (ICU and burn patients); dissolves fibrin clot. proximity to patients with MRSA; after receiving broad spectrum LIPASE (FAT-SPLITTING ENZYME): produced by both coagulase- antibiotics; nasal carriage. positive and coagulase-negative staphylococci. It is important for the o It may also be resistant to other semisynthetic penicillin formation of furuncles, carbuncles, and boils. Staphylococcus epidermidis DEOXYRIBONUCLEASE (DNAse) AND PHOSPHATASE: it lowers the o Normal flora of skin viscosity of exudates, giving the pathogen more mobility. o Contaminant of medical instruments – catheters, CSF shunts and BETA-LACTAMASE: breakdowns penicillin and other beta-lactam drugs. prosthetic heart valve implants ENTEROTOXINS (HEAT-STABLE): these toxins appear to act as o Secretes poly-gamma-D-L-glutamic acid which provides adherence neurotoxins that stimulate vomiting through the vagus nerve to devices o Produced by 30-50% of isolates. These are superantigens (like o Has been known to cause various hospital-acquired infections TSST-1) that have the ability to interact with many T cells, activating o Culture: small to medium-sized non-hemolytic, nonpigmented, an aggressive immune response. white opaque, pin-head colonies (BAP) o Example: Enterotoxins A, B, and C: responsible for food o Biochemical test: MSA (-) coagulase-negative staphylococci poisoning (the infected food handler is the source of contamination) (CoNS) o Enterotoxin B: associated with pseudomembranous enterocolitis o Antimicrobial test: susceptible with 5 µg Novobiocin (16-27 mm) LEUKOCIDIN / PANTON-VALENTINE LEUKOCIDIN (CYTOLYTIC Staphylococcus saprophyticus TOXIN): it attacks and kills WBC (PMN, macrophages, and monocytes) o Associated with community-acquired UTI in young, sexually active o Pore-forming exotoxin that kills WBC; it suppresses phagocytosis females. o Responsible for necrotizing skin and soft tissue infections o Adheres more effectively to the epithelial cells lining the urogenital HEMOLYSIN (CYTOLYTIC TOXIN): it causes anemia-make iron tract than other CoNS available for microbial growth o Rarely found on other mucous membranes or skin surfaces o ALPHA HEMOLYSIN: it destroys RBC, platelets, and o Culture: white opaque, pin-head, slightly large colonies; 50% of the macrophages. It causes severe tissue damage strains produced yellow pigment; nonhemolytic (BAP) o BETA HEMOLYSIN (hot-cold lysin / spingomyelinase C): it has o Biochemical tests: MSA (-/+); coagulase-negative staphylococci; enhanced hemolytic activity with incubation on 37ºC and subsequent DNAse (-) exposure to 4ºC (“hot-cold” lysin) o Urine culture: 10,000 CFU/mL (significant findings) o GAMMA HEMOLYSIN: less toxic as compared to alpha and beta o Antimicrobial test: Resistant to 5 µg Novobiocin (6-12 mm zone of lysins inhibition) o DELTA HEMOLYSIN: it destroys RBC, and it is associated with the Staphylococcus lugdunensis Panton-Valentine Leukocidin o Coagulase-negative staphylococci by tube method EXFOLIATIN A AND B (SUPERANTIGEN) / EPIDERMOLYTIC TOXIN A o More aggressive than the other CoNS in its ability to be infective – AND B causes the epidermal layer of the skin to slough off-stratum associated with catheter-related bacteremia and endocarditis granulosum. It causes scalded skin-syndrome (SSS) or Reiter disease. o Contains the mecA gene which codes for oxacillin resistance TOXIC SHOCK SYNDROME A (TSST-1) / ENTEROTOXIN F / PYROGENIC EXOTOXIN: It is a chromosomal-mediated toxin. It causes NOVOBIOCIN SUSCEPTIBILITY almost all cases of menstruating-associated TSS. o It stimulates production of a large number of cytokines that are PURPOSE: to differentiate S. saprophyticus (resistant) from other CoNS responsible for the symptoms. which are sensitive to novobiocin. PROTEIN A: it is antiphagocytic by competing with neutrophils for the Fc PRINCIPLE: After incubation with 5 µg of novobiocin, S. saprophyticus is portion of specific opsonin not inhibited by antibiotic other CoNS are susceptible INTERPRETATION: DIFFERENTIAL TESTS FOR STAPHYLOCOCCUS AUREUS o (S): zone diameter >16 mm o (R): zone diameter ≤ 16 mm COAGULASE TEST: it is the best single criterion of pathogenicity of Staphylococcus aureus LABORATORY DIAGNOSIS o Reagent: Rabbit plasma o Slide Method: used to screen catalase-positive colonies SPECIMENS: aspirated secretions (best samples), purulent exudates, ▪ It detects cell bound coagulase / clumping factor on the surface and joint fluids of the cell wall GRAM STAIN: gram-positive spherical cells that appear singly, in pairs, and in clusters.  CULTURE: Culture media is BAP, MSA, PEA, CAN, CAP, BHI, and ▪ Species: Streptococcus pyogenes, groups A, C, and G thioglycolate streptococci (large-colony-forming-isolates) o MSA, CAN, and PEA: used for heavily contaminated specimens o Viridans group CATALASE TEST ▪ It will grow both 45ºC and 37ºC o Reagent: 3% H2O2 ▪ It is not part of the Lancefield group; may be alpha hemolytic or o (+) result: bubble formation / effervescence non-hemolytic (gamma hemolytic) o The colonies which will be used for this test should not be taken ▪ Normal biota in the urinary tract (URT) in humans from BAP because of the presence of peroxidase in that medium ▪ Some may have A, C, G, or N antigens COAGULASE TEST ▪ Species: Streptococcus salivarius, Streptococcus mutans, o Reagent: rabbit plasma Streptococcus mitis o (+) result: clot / coagulum formation o Lactic group o Isolates those that do not produce either clumping factor or ▪ It will grow on 10ºC and 37ºC staphylocoagulase are reported as coagulase-negative staphylococci ▪ Non-hemolytic organisms with Lancefield group N antigen MANNITOL FERMENTATION TEST ▪ Often found in dairy products o (+) yellow halo around colonies of mannitol fermenting- ▪ Species: Streptococcus lactis – cause normal coagulation or staphylococci souring of milk DNAse TEST o Enterococcus group o 0.1N HCl: agglutinate or precipitate protein ▪ It will grow at 10ºC, 45ºC, and 37ºC o 0.1% toluidine blue: (+) pink color (cell died); (-) blue color (cell is ▪ Normal flora of human intestine alive) ▪ Species: Enterococcus faecalis PYRROLIDONYL ARYLAMIDASE TEST SMITH AND BROWN CLASSIFICATION o It differentiates the coagulase-positive Staphylococci o Substrate: pyroglutamyl-β-naphthylamide (L-pyrrolidonyl- β- It is based on hemolytic patterns naphthylamide) o Alpha-hemolytic Streptococci o Final reagent: p-dimethylaminocinnamaldehyde ▪ They have partial / incomplete hemolysis of red blood cells o End product: L-pyrrolidone and B-naphthylamide (red color) around colony. o (+) result: S. lugdunensis, S. intermedius, S. schleiferi ▪ Green hemolysis / discoloration around colony. o (-) result: S. aureus ▪ Species: Streptococcus pneumoniae (green streptococci) o Beta-hemolytic Streptococci ▪ They exhibit complete lysis of red blood cells around colony ▪ Clear area / zone around colony ▪ Species: Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysagalactiae subsp., Equisimilis and Streptococcus angionosus group. o Gamma-hemolytic Streptococci / Non-hemolytic Streptococci ▪ No lysis of red blood cells around colony / red cells immediately surrounding the colony are unaffected (no change) ▪ Species: Enterococcus faecalis LANCEFIELD CLASSIFICATION (ANTIGEN SEROGROUPING) It is based on the extraction of C carbohydrate from the Streptococcal cell wall Rebecca Lancefield – found out that the C carbohydrate can be extracted from the streptococcal cell wall by placing the organisms in dilute acid heating for 10 minutes Mostly significant in classifying β-hemolytic streptococci Hemolytic Reaction Lancefield Group Species β-hemolytic A S. pyogenes B S. agalactiae C S. dysagalactiae subsp., Equisimus, S. equi α, γ D S. bovis group β, α, or γ D Enterococci α, β, or γ A,C,F,G,N or none Viridans streptococci α None S. pneumoniae GRAM-POSITIVE COCCI STREPTOCOCCACEAE GROUP A STREPTOCOCCI STREPTOCOCCI It is not considered as part of the normal flora – pathogenic to man Commonly found as part of normal human flora, however, when these It is acquired through contaminated droplets by cough or sneeze organisms gain access to normally sterile sites, they can cause life It is resistant to drying and can be isolated from swabs after several hours threatening infection. of collection. All streptococci except the viridans group have a layer of C-carbohydrate Species: Streptococcus pyogenes – “fever producing bacteria,” flesh for serological classification. eating bacteria Notorious pathogens: S. pyogenes and S. pneumoniae Culture: small, translucent and smooth; well-defined β-hemolysis Aerococcus, Lactobacillus, Leuconostoc, and Pediococcus – Principal virulence factor: M-protein resemble streptococci Other virulence factors: Microscopy: Gram-positive, spherical, arranged in chains or pairs; young o Protein F: mediates epithelial cell attachment cultures are characterized by the presence of capsules. o Lipoteichoic acid: bacterial adherence to the respiratory epithelium Culture: grayish, pinpoint, translucent to slightly opaque colonies; mucoid o Hyaluronic acid capsule: weakly immunogenic; prevents opsonized colonies; growth is enhanced by blood, serum, or glucose incorporated in phagocytosis; masks its antigen agar plate. o Hemolysins Biochemical tests: Catalase (-); oxidase (-); gas production (-); non- o Toxins motile; ferments carbohydrate o Enzymes ACADEMIC / BERGEY’S CLASSIFICATION HEMOLYSINS It is based on temperature requirement. Streptolysin O: “oxygen labile;” highly antigenic, responsible for o Pyogenic group subsurface hemolysis on BAP incubated anaerobically – it is best to stab ▪ It will neither grow on 10ºC or 45ºC, only at 37ºC the agar to create anaerobiosis ▪ Produce pus, mostly β-hemolytic o Also cause lysis of WBCs, platelets, tissue cells; induces antibody response  o Anti-streptolysin O test – detect recent with S. pyogenes (4-fold STREPTOCOCCAL TSS titer) Streptolysin S: “oxygen stable;” nonantigenic, responsible for surface It is a condition in which the entire organ system shuts down, leading to hemolysis on BAP incubated aerobically death. o Also cause lysis of WBCs The initial strep infection includes pharyngitis, cellulitis, and wound infections DEOXYRIBONUCLEASES (DNAse) SpeA play a major role in the pathogenesis of this disease 4 types: A, B, C, and D (antigenic enzymes) LABORATORY DIAGNOSTIC TESTS FOR GROUP A STREPTOCOCCI It lowers viscosity of exudates, giving the pathogens more mobility The antibodies to these enzymes can be detected following infections BACITRACIN DISC TEXT / TAXO A (0.04 UNITS) o To differentiate between S. pyogenes from other β-hemolytic STREPTOKINASE groups. o It is useful in screening for group A streptococci in throat cultures It causes lysis of fibrin clots o (+) result: any zone of inhibition (susceptible) Protein that binds to plasminogen and activates the production of plasmin o Groups C and G are also susceptible to bacitracin Allows the bacteria to move from the clotted area – spread of infection SULFAMETHOXAZONE AND TRIMETHOPRIM (SXT) TEST Activates a host blood-factor that dissolves fibrin clots o (+) result: resistant (groups A and B) o Most interfering respiratory microbiota will be inhibited by SXT HYALURODINASE (SPREADING-FACTOR ENZYME) o Group C Streptococci is susceptible (negative) to SXT L-PYRIROLIDONYL-A-NAPHTHYLAMIDE (PYR) TEST It solubilizes the ground substance of mammalian tissue o Specific than bacitracin test. To separate the tissue and spread the organism o Detects the presence of PYRase or pyrolidonyl-arylamide enzyme o S. pyogenes (+) PYR test PYROGENIC (SPES) TOXINS – SEROTYPES A, B, C (SUPER ANTIGENS) o (+) result: bright / cherry red color upon adding 0.01% of cinnamaldehyde reagent (p-dimethyl-aminocinnamaldehyde) Formerly known as erythrogenic toxins o (-) result: no color change on the paper disk Exotoxin B (cysteine protease) – degrades proteins; mediates rash in o Other PYR (+) test organisms: Enterococcus, Aerococcus, scarlet fever Gemella INFECTIONS AND DISEASES GROUP B STREPTOCOCCI Pharyngitis or tonsilitis (Strep throat) Part of the normal flora of female genital tract and lower gastrointestinal o Spread by droplets and close contact tract. o When highly virulent strains appear in schools, they can cause It is nosocomially transmitted by unwashed hands of mother or health sharp outbreaks of sore throats and scarlet fever care personnel to the newborn or infant. o Diagnosis relies on a throat culture or direct antigen detection It causes infections of fetus (infection during passage through the Scarlet fever (scarlatina) colonized birth canal and premature rupture of mother’s membrane) o A punctuate exanthem overlying erythema and appears initially on It is recommended that all pregnant women be screened for group B the neck and upper chest. 1-2 days following strep throat streptococci at 35-37 weeks of gestation. Lysogenic bacteriophage (T12) – pyrogenic exotoxins Species: Streptococcus agalactiae Cardinal signs: diffused red rash on the upper chest and spreads to the Culture: grayish-white, mucoid colonies with small zone of beta- trunk and extremities, and strawberry-colored tongue. hemolysis Susceptibility test: Dick’s test (erythrogenic toxin) Virulence factor: capsule (sialic acid) o (+) result: erythema or redness of the test site Avirulent factors: hemolysin, CAMP factor, neuramidase, Diagnostic test (current infection): Schultz Charlton (anti-erythrogenic deoxyribonuclease, hyalurodinase, and protease. toxin) o (+) result: “blanching phenomenon” – rash fade INFECTIONS OR DISEASES SKIN INFECTIONS (CELLULITIS, ERYSIPELAS, IMPETIGO) Pneumonia Meningitis Cellulitis: it is a diffuse, spreading infection of subcutaneous skin tissue Neonatal sepsis characterized by a defined area of redness and accumulation of fluid. Postpartum infection Erysipelas: an acute infection and inflammation of the dermal layer of the Osteomyelitis skin characterized by painful reddish patches that enlarge and thicken with Urinary tract infection a sharply defined edge. Myocarditis Skin infection caused by a group A streptococci strains may also lead to necrotizing fasciitis (“galloping gangrene or flesh-eating bacteria LABORATORY DIAGNOSTIC TESTS FOR GROUP B STREPTOCOCCI syndrome) Necrotizing fasciitis (NF) may be categorized as type 1, 2, and 3. CAMP TEST Type 2 NF consists of only group A streptococci; type 3 NF is gas o To different S. galactiae from other β-hemolytic streptococci gangrene and clostridial myonecrosis o It is performed in an ambient gas at 35-37ºC o CAMP Factor – enhances β-hemolytic activity; extracellular; RHEUMATIC FEVER thermostable antigen o Reagent: β-lysin producing strain of S. aureus or β-lysin disk It is characterized by fever, inflammation of the heart, joint, and blood o (+) result: arrow-head - β-hemolysis near S. aureus growth or (+) vessels. It is a complication of pharyngitis. bowtie appearance HIPPURATE HYDROLYSIS TEST ACUTE GLOMERULONEPHRITIS (BRIGHT’S DISEASE) o To differentiate S. agalactiae from other β-hemolytic streptococci o It is performed in an ambient gas at 35-37ºC It is an inflammatory disease of the renal glomeruli; results from the o S. agalactiae possesses the enzyme hippuricase or Hippurate deposition of antigen-antibody complexes; complication of pharyngitis. hydrolase o Reagents: Sodium Hippurate + ninhydrin o Results: (+) purple color after adding Ninhydrin reagent (indicate Hippurate hydrolysis); (-) no color change GROUP C AND G STREPTOCOCCI These organisms are recovered from urinary tract, vagina, skin of humans. They possess M protein just like Group A Streptococci They are the main source of streptokinase, an animal pathogen  Species: S. dysagalactiae subsp., Equisimilus, S. equi subsp. BILE SOLUBILITY TEST (CONFIRMATORY TEST) Zoopidemicus o This test evaluates the ability of S. pneumoniae to lyse in the Antimicrobial susceptibility tests: presence of bile salts o Group C Streptococci is susceptible to bacitracin and SXT. o Differentiates pneumococcus from viridans streptococci o Group G Streptococci may be bacitracin resistant or susceptible. o Reagent: Sodium desoxycholate (bile salt) o When a heavy suspension of pneumococcus is added to bile salt, VIRIDANS STREPTOCOCCI the cloudiness of the broth clears after 3 hours of incubation o Result: (+) bile soluble, (-) turbid Are oropharyngeal commensals o Quellung reaction: form the basis of serotyping and depends on Are opportunistic pathogen of low virulence, fastidious bacteria the swelling of capsule upon binding of homologous antibody Also known as alpha-prime streptococci that lack Lancefield group SKIN TEST (FRANCIS TEST) antigens o To detect the presence of antibodies against pneumococci Infections: SBE, gingivitis, dental carries, meningitis and osteomyelitis CO-AGGLUTINATION TEST Laboratory tests: bile insoluble, optochin resistant, penicillin test (s), no o Uses antibody to a particle to enhance visibility of the agglutination growth in 6.5% NaCl, LAP (+) and PYR (-) reaction between antigen and antibody LABORATORY TESTS FOR THE S. BOVIS GROUP (GROUP D ENTEROCOCCI STREPTOCOCCI / NONENTEROCOCCI Belong to family Streptococcaceae GROWTH IN BILE ESCULIN MEDIUM Formerly known as Group D Streptococci o Esculin hydrolysis tests for the cleavage of a glycoside Natural inhabitants of intestinal tracts of humans and animals o Reagent: esculin + 1-4% bile salt They exhibit pseudocatalase reaction (weak bubbling in catalase test) o (+) result: black color complex in the agar within 48 hours Resistant to multiple antimicrobial agents 6.5% NaCl (NUTRIENT BROTH BASE) TEST Hemolytic patterns may be α, β, or nonhemolytic o Result: no growth / negative Virulence factors: extracellular serine protease, gelatinase, and cytolysin DIFFERENCE BETWEEN ENTEROCOCCI AND NON-ENTEROCOCCI Species: E. faecalis, E. faecium, E. avium, E. gallnarum, E. durans, E. Organism Bile Esculin Growth with 6.5% PYR test NaCl raffinosus Enterococci + + + Related infections: UTI, endocarditis, bacteromia, wound infection Non-enterococci + - - ENTEROCOCCI IDENTIFICATION STREPTOCOCCUS PNEUMONIAE Enterococci are identified based on their ability to: Also known as diplococcus / pneumococcus o Produce acid in carbohydrate broth Considered part of the normal flora of the URT of preschool children o Produce acid from methyl-α-glucopyranoside (MGP) Causative agent of lobar pneumonia – most common cause of bacterial o Hydrolyze arginine pneumonia in elderly as well as in patients with underlying disease o Tolerate 0.04% tellurite The cell wall contains an antigen referred to as C substance, which is o Utilize pyruvate similar to the C carbohydrate of the various Lancefield groups. o Growth around 100-µg efromycin acid disk Microscopy: Gram-positive cocci in pairs, oval or lancet shape facultatively anaerobe LABORATORY TESTS FOR ENTEROCOCCI Culture: alpha-hemolytic colonies; mucoid, flat colonies with depressed Specimens: Blood, urine, wound secretions center o GRAM STAIN Principal virulence factor: capsular polysaccharide ▪ Gram-positive cocci in pairs and long chains Avirulent factors: hemolysin, IgA protease, neuraminidase, and o CULTURE hyaluronidase ▪ Culture media: TSB or BHI with 5% sheep’s blood (routinely used) INFECTIONS / DISEASES ASSOCIATED ▪ Selective media for contaminated specimens: BE azide agar, CAN agar, PEA and Cephalexin-Aztreonam-Arabinose agar Lobar Pneumonia: individuals with this infection show bloody, rust-tinged ▪ Enterococci grows well at 35ºC with CO2 sputum, large numbers of WBC in sputum and predominance of S. ▪ E. faecalis is easily detected by its ability to grow in medium pneumoniae. containing tellurite. o Pneumonia due to S. pneumoniae is not usually a primary infection o GROWTH IN BILE ESCULIN (BE) MEDIUM but rather a result of disturbance of normal defense barriers ▪ It is a two-step test detecting to growth of bacteria in the Meningitis (infants / elderly) presence of 40% bile and its ability to hydrolyze esculin Otitis media ▪ Reagent: esculin ÷ 1% to 4% bile salt Bacteremia ▪ (+) result: black color complex agar within 48 hours Endocarditis o 6.5 NaCl (NUTRIENT BROTH BASE) TEST Peritonitis ▪ (+) result: TURBIDITY: Enterococci (also PYR+) ▪ (-) result: Non-enterococci LABORATORY DIAGNOSIS OF STREPTOCOCCUS PNEUMONIAE Specimen: sputum, blood, and CSF. o A rust-tinged sputum may indicate S. pneumoniae. GRAM STAIN o The ends of the cells are slightly pointed (oval or lancet-shaped), appear in pairs or short chains. o Direct smears of CSF reveal WBCs and Gram-positive cocci in pairs CULTURE o Culture media: BHI, trypticase soy agar with 5% sheep’s blood, CAP o Young colonies: “dome-shaped,” glistening, wet and mucoid o Old colonies: “coin with a raised rim,” dimple-shaped / donut- shaped colonies o Clinical isolates and stock cultures require frequent subculturing to ensure viability and prevent autolysis OPTOCHIN DISK TEST / TAXO P o Susceptibility medium: BAP o Ethyldroxycuprein hydrochloride (6 µm to 10 µm disk) o Susceptibility test