Bacteriology Lecture Notes - Gram Positive Cocci PDF

BACTERIOLOGY GRAM POSITIVE COCCI LECTURE STAPHYLOCOCCI Gram (+) cocci in grape like clusters; non-motile Belongs to the Family Micrococcaceae (Staphylocci and Micrococci) Facultative anaerobes EXCEPT Staphylococcus saccharolyticus Able to grow in 7.5-10% NaCl Resistant to 0.04 unit of Bacitraci...

BACTERIOLOGY GRAM POSITIVE COCCI LECTURE STAPHYLOCOCCI Gram (+) cocci in grape like clusters; non-motile Belongs to the Family Micrococcaceae (Staphylocci and Micrococci) Facultative anaerobes EXCEPT Staphylococcus saccharolyticus Able to grow in 7.5-10% NaCl Resistant to 0.04 unit of Bacitracin Beta hemolytic on BAP CATALASE: A biochemical test used to separate Staphylococci (+), Micrococci (+) and Streptococci (-) (+) Result: vigorous bubbling, uses Hydrogen peroxide Do not use colonies from BAP (False Positive Reaction) Staphylococcus aureus General Characteristics Aureus means "GOLD"; MOST VIRULENT SPECIES Normal flora of anterior nares and nasopharynx Facultative anaerobes PCR will enable detection from nasal swab Growth/Colony Morphology Uniform turbidity on broth Grow in BAP as creamy, white with pin-head colonies On plates, they produce smooth circular and opaque colonies with oil like butyrous appearance JET BLACK colonies on tellurite agar/Vogel Johnson Medium Tests for Identification 1. COAGULASE TEST DEFINITIVE TEST FOR S. aureus Test to differentiate from other Staphylococci A. SLIDE COAGULASE (SCREENING TEST) Detects: cell bounded coagulase / clumping factor Reagent: rabbit plasma (must be obtained using EDTA top tube) (+) result: CLUMPING B. TUBE COAGULASE (CONFIRMATORY TEST) Detects: free coagulase Reagent: rabbit plasma (must be obtained using EDTA top tube) (+) result: CLOT 2. MANNITOL FERMENTATION TEST Media: Inhibitor: pH indicator: (+) Result: MSA (1% Mannitol) 7.5% NaCl PHENOL RED YELLOW 3. DNASE TEST aka Thermonuclease Test detects deoxyribonuclease A. DYE METHOD DNA + Methyl Green = Clear Zone around the colonies DNA + 0.1% Toluidine Blue = Pink Zone around the colonies (+) Pink Zone = Cell is DIED (-) Blue Color - Cell is ALIVE B. HCI PRECIPITATION METHOD (+) Result Clearing of Agar around the colonies Bacteria which are DNase test (+) Serratia marcescens, Moraxella catarrhalis, Aeromonas, Streptococcus pyogenes, Helicobacter pylori, Vibrio cholerae, Stenomaltophilia, Staphylococcus aureus 4. LATEX AGGLUTINATION FOR PROTEIN A Antigen detection S. aureus with protein A as inert particles to which antibody (Fc fragment) binds Detects specific bacterial antigens S. pneumoniae, N. meningitidis, N.gonorrhea, H. influenzae Virulence Factors 1. LIPASE a fat splitting enzyme; associated with SKIN INFECTIONS such as boils, carbuncles, furuncles 2. HYALURONIDASE aka T FACTOR or the DURAN-RAYNAL FACTOR a SPREADING FACTOR that enhances invasion to tissue; causes hydrolysis of hyaluronic acid 3. BETA LACTAMASE makes S. aureus resistant to penicillin hydrolysis of penicillin 4. STAPHYLOKINASE aka FIBRINOLYSIN dissolves fibrin clot and may enable infection to spread once clot is dissolved. 5. COAGULASE causes bacteria to agglutinate in plasma; converts fibrinogen to fibrin; may coat neutrophils with fibrin formed to protect organisms from PHAGOCYTOSIS 6. DNASE aka Thermonuclease causes degradation of DNA; decrease viscosity of exudate giving organisms more mobility 7. BETA HEMOLYSIN causes destruction of RBC responsible for beta-hemolysis of S. aureus; ANEMIA 8. PROTEIN A prevents phagocytosis has the ability to bind FC portion of IgG found on the CELL WALL 9. GELATINASE Gelatin ---> Amino acid End product: amino acid refrigerator temperature (+) gel will liquefy; (-) gel will solidify 6. DNASE aka Thermonuclease causes degradation of DNA; decrease viscosity of exudate giving organisms more mobility 7. BETA HEMOLYSIN causes destruction of RBC responsible for beta-hemolysis of S. aureus; ANEMIA TOXINS: 1. ENTEROTOXIN group of seven heat stable protein (A, B, C, C2, D, E, F). Most staphylococcal related food poisonings are associated with Enterotoxin A and B. They are heat-stable (stable at 100 degC for 30 minutes) exotoxins that cause diarrhea and vomiting A. Enterotoxin A and B Food Poisoning (Heat Stable) B. Enterotoxin F/TSST Toxic Shock Syndrome Toxin 1 (TSST-1) most cases are in menstruating women, particularly using tampons. 2 EXFOLIATIN A AND B aka EPIDERMOLYTIC TOXIN A and B; hydrolyze tissue through cleavage of STRATUM GRANULOSUM; associated with staphylococcal SCALDED SKIN SYNDROME aka Ritter Disease. 3. PHANTON-VALENTIN LEUKOCIDIN destroys WBC involved community acquired MRSA; inhibits phagocytosis May be mistaken for S. aureus 1. Staphylococcus lugdunensis Slide coagulase (+) but Tube Coagulase (-) Contains mec-A gene (for Oxacillin resistance) PYR (+) Narrow beta hemolytic Mannitol (+) Catheter-related Endocarditis and Bacteremia 2. Staphylococcus intermedius Slide coagulase (+) PYR (+) and VP (-) A dog Pathogen 3. Staphylococcus haemolyticus Coagulase (-) Beta hemolytic Coagulase Negative Staphylococci S. epidermidis OTHERS Normal skin flora Causes UTI, stitch abscess PROSTHETIC HEART VALVE INFECTION ability for slime; production or biofilm formation BB Contaminant at RT Slime or POLY-GAMMA-DLGLUTAMIC ACID VIRULENCE FACTORS enhances attachment of organisms to plastic catheters S. saphrophyticus Most common cause of UTI among sexually active young woman Causes pyelonephritis and cystitis in those with indwelling catheters >10,000 CFU/mL - considered significant UNCERTAIN MICROCOCCI Gram (+) cocci in TETRADS STRICT AEROBES BAP - GAMMA Hemolytic; white/yellow pigments It grows on FUROXONE-TWEEN-80 OIL RED O AGAR Modified Oxidase Test PRESUMPTIVE TEST for Micrococci Uses tetramethyl p-phenylenediamine dihydrochloride in (DMSO) dimethylsulfoxide (+) Result: BLUE COLOR within 2min Tests to differentiate Staphylococcus and Micrococci Staphylococus Micrococcus Aerobic Growth Positive Positive Anaerobic Growth Positive Negative Catalase test Positive Positive Modified Oxidase Test Negative Positive 0.04 u Bacitracin Resistant Susceptible Furazolidone/Furoxone Susceptible Resistant Lyostaphin Susceptibility Susceptible Resistant Benzidine Test Negative Positive Glucose Utilization of Medium Fermenter Oxidizer STREPTOCOCCI Gram positive cocci in chains, spherical to ovoid; Non-motile; FACULTATIVE ANAEROBES, some are CAPNOPHILIC All streptococci EXCEPT Viridans group and S. pneumonia have a layer of -carbohydrate for serological classification. Non-pigmented, pinpoint colonies, CATALASE (-) Smith and Brown Classification ALPHA-HEMOLYTIC STREPTOCOCCI causes PARTIAL/INCOMPLETE hemolysis on BAP S. pneumoniae Viridans Strep S. mitis S. mutans BETA-HEMOLYTIC STREPTOCOCCI GAMMA-HEMOLYTIC STREPTOCOCCI causes COMPLETE hemolysis on BAPproduces CLEAR ZONES around the colonies UNABLE to cause hemolysis red with colonies S. pyogenes S. agalactiae E. faecalis E. faecium S. bovis Lancefield Classification based on the extraction of Carbohydrate from Streptococcal CELL WALL All Streptococci EXCEPT Viridans and S. pneumonia are placed under Lancefield GROUP A Streptococcus pyogenes GROUP B Streptococcus agalactiae GROUP C GROUP D Streptococcus zooepidemicus Streptococcus equi Streptococcus equisimilis Streptococcus dysaggalactiae Streptococcus bovis Streptococcus equinis Enterococcus faecalis Enterococcus faecium Enterococcus avium Enterococcus durans GROUP A: S. pyogenes Growth/Colony Morphology Best isolated on BAP, addition of SXT can enhance its recovery from heavily contaminated specimens Pathogenic to Man (Not part of the normal flora) Test for Identification Susceptible to bacitracin (TAXO A) (0.02-0.04 units) Susceptible to Penicillin (Drug of Choice for Strep) Resistant to SXT PYR (+) Catalase (-) VIRULENCE FACTORS: 1. Streptolysin STREPTOLYSIN-O STREPTOLYSIN-S Hemolysis Type SUB-Surface Hemolysis Surface Hemolysis Characteristics Oxygen-labile Can cause hemolysis when incubated ANAEROBICALLY Oxygen-Stable Can cause hemolysis when incubated AEROBICALLY Antigenicity Immunogenic Can stimulate production of antibodies Non-immunogenic Cannot stimulate production of antibodies Effect Lysis of WBC, platelets, tissue cells Lysis of WBC 2. Erythrogenic Toxin/Pyrogenic Toxin A, B, C (Superantigens) causes fever, alteration of blood brain barrier may be associated with organ damage and SKIN RASHES of SCARLET FEVER 3. EXOTOXIN B (Cysteine Protease) degrades proteins mediates RASH in SCARLET FEVER 4. Hyaluronidase Spreading factor solubilizes the ground substances of mammalian tissue 5. Streptokinase fibrinolysin that lyses blood clots prevents fibrin barrier and allows spread of infection 6. M-Protein MAJOR VIRULENCE FACTOR prevents Phagocytosis and interferes with complement activity 7. Protein F enhances attachment to epithelial cell 8. Streptodornase/DNAse group of 4 antigenic enzymes (A,B,C,D) with nuclease activity degrade host DNA and/or RNA It lowers the viscosity of exudates, giving the pathogens more mobility DISEASES 1. STREP THROAT spread by droplets and close contact 2. SCARLET FEVER form of pharyngitis with rashes with "strawberry tongue" 3. POST SEQUELAE repeated episodes of pharyngitis 4. Acute Glomerulonephritis (AGN) or Bright's Disease inflammatory disease of the renal glomeruli; results from deposition of antigenantibody complexes; complication of PHARYNGITIS 5. Rheumatic Fever characterized by fever, inflammation of the heart, joints and blood vessels; it is also a complication of PHARYNGITIS DIAGNOSTIC TESTS: DICK’S TEST a susceptibility test for SCARLET FEVER (+) Result: REDNESS at the site of infection Schultz-Charlton Test/ Blanche Phenomenona suscep To determine if the rashes is caused by SCARLET FEVER or NOT It involves injection of anti-erythrogenic toxin (+) Result: Fading of rashes 6. Streptococcal TSS is a condition in which the ENTIRE ORGAN SYSTEM shuts down, leading to death. The initial strep infections include pharyngitis, cellulitis and wound infection 7. Skin Infections: A. Erysipelas inflammation of the DERMAL LAYER of the skin with PAINFUL REDDSIH patches B. Cellulitis subcutaneous skin tissue characterized by a defined area of redness (erythema) and accumulation of fluid (edema) C. Impetigo A. Necrotizing fasciitis aka Galloping Gangrene or Flesh-eating bacteria Syndrome - a rapidly progressing skin infection Necrotizing Fasciitis may be categorized as Type 1, 2, or 3 Type 2 NF consists of only GROUP A Streptococci Type 3 NF is a GAS GANGRENE and clostrdial myonecrosis GROUP B: S. agalactiae Growth/Colony Morphology Normal flora of the GIT, pharynx and vaginal tract It is nosocomially transmitted by unwashed hands of mother or health care personnel to the newborn or infants It is recommended that all pregnant women be screened for Group B streptococci at 35-37 weeks gestation Culture: Grayish white, mucoid colonies with small zone of beta- hemolysis Virulence Factors Capsule (Sialic acid) significant component of the capsule) Virulent Factor Hemolysis, CAMP Factor Neuraminidase DNAse, Hyaluronidase and Protease Tests for Presumptive ID A. CAMP TEST Positive for S. agalactiae CAMP Factor - enhances BETA-HEMOLYTIC Activity; extracellular, thermostable antigen Media: BAP Known Organism: S. aureus (beta-hemolytic) (+) Result: Arrow Head Zone of beta-hemolysis (Enhanced Hemolysis) or BOWTIE appearance (-) Result: No Enhanced Hemolysis B. HIPPURATE HYDROLYSIS detects hydrolysis of SODIUM HIPPURATE to BENZOIC ACID and GLYCINE (to be detected). the positive result is due to enzyme: HIPPURICASE/HIPPURATE HYDROLASE (+) Result: Purple Reagent: Ninhydrin C. LIM BROTH, GRANADA may be used for Group B Streptococci D. BACITRACIN and SXT RESISTANT Diseases A. Neonatal Meningitis transmission to the infant may occur during delivery B. Pneumonia C. Bacteremia leading cause of neonatal sepsis D. Osteomyelitis, Postpartum infection, UTI and Endocarditis leading cause of neonatal sepsis GROUP C Streptocci Growth/Colony Morphology These are ANIMAL PATHOGEN, may cause severe pharyngitis followed by bacteremia, may also cause pneumonia, cellulites and abscess (S. dysagalactiae subsp. equisimulus, S. equi subsp. zooepidemicus) GROUP C Strep are the MAIN SOURCE of STREPTOKINASE GROUP C and G Strep possess M protein just like Streptoccus pyogenes Beta-hemolytic, Bacitracin (Resistant) and SXT (Sensitive) Group F - S. anginosus: causes cellulites Group G - usually found as normal flora of human GIT, vaginal tract and oropharynx GROUP D Streptocci Growth/Colony Morphology BILE ESCULIN: PRESUMPTIVE TEST FOR GROUP D Uses 40% Bile; Media: BILE ESCULIN MEDIA (BEA) -slant Reagent/Indicator: Ferric Ammonium Citrate (+) Result: blackening of medium Differential test for Group D Non-enterococci and Enterococci Enterococci Non-Enterococci Bile Esculin (+) (+) Salt Tolerance Test (+) (-) Penicillin Disk Test R S PYR (+) (-) Growth at 45 deg( (+) (+) Growth at 10 degC (+) (-) Enterococci Normal GUT, GIT and RT Flora Grows in 6.5% NaCl Resistant: Penicillin Cephalosporin Vancomycin Bacitracin Positive Bile Esculin PYR Tests Hippurate hydrolysis Most commonly isolated from human infection E. faecalis E. faecium NOSOCOMIAL UTI Growth at 45 degC and 10 degC Group D Streptococci (GROUP D Non-Enterococci)/ S. bovis group S. bovis and S. equines Grows inhibited at 6.5% NaCl Penicillin Sensitive; Vancomycin Resistant Bile Esculin(+) PYR (-) Causes UTI, endocarditis and septicemia Isolation of this in the blood may be indicative of "COLON CANCER" 45 degC (+); 10 degC (-) Streptococcus pneumoniae General Characteristics Normal Flora of Nasopharynx and oropharynx (URT) of PRESCHOOL children The cell wall contains an antigen referred to as C SUBSTANCE, which is similar to the C CARBOHYDRATE of the various Lancefield Group Virulence Factor Capsular Polysaccharide composed of HYALURONIC ACID Avirulent Factors Hemolysin Immunoglobulin A Protease Neuraminidase and Hyaluronidase Morphology GRAM (+) cocci in pairs, oval or lancet or bullet shaped diplococci; FACULTATIVELY ANAEROBE Growth/Colony Morphology and Growth Requirements Requires 5-10% CO2 CULTURE: alpha-hemolytic colonies; mucoid flat colonies with depressed center Young colonies are elevated or "DOME-SHAPED" (24hrs) Age or old colonies assumes the so-called Nail-head/Crater-like appearance (48hrs) Tests for Identification A. Bile Solubility Test CONFIRMATORY Test Used to differentiate Pneumococcus from Viridans Strep Media: BAP Culture Media: Add 10% Sodium desoxycholate (+) Result: LYSIS OF COLONIES (-) Result: INTACT COLONIES Broth: Add 2% Sodium desoxycholate (+) Result: CLEARING OF MEDIA (-) Result: Remain turbid B. Optochin Test Susceptible: S. pneumoniae (>14mm zone) PRESUMPTIVE Test Test that will differentiate Pneumococci from other alpha-hemolytic Streptococci uses TAXO P Chemical Name: ETHYLENEHYDROCUPREINE HYDROCHLORIDE 10 ug: >14 mm; 6 ug: 10 mm C. Capsular Swelling (+) D. FRANCIS TESTING - skin test for pneumonia DISEASES: #1 cause of ADULT BACTERIAL Meningitis MOST COMMON AGENT OF OTITIS MEDIA Lobar pneumonia, associated with RUSTY or ANCHOVY SAUCE DIFFERENTIAL TEST FOR PNEUMOCOCCI & VIRIDANS STREP. PNEUMOCOCCI VIRIDANS STREP. Mouse Virulence (+) (-) Inulin Fermentation (+) (-) Bile Solubility (+) (-) Optochin S R Neufeld-Quellung (+) (-) VIRIDANS STREPTOCOCCI Oropharyngeal commensal also known as ALPHA-PRIME Streptococci that LACK the LANCEFIELD GROUP ANTIGEN MOST COMMON CAUSE OF SBE Individual with Rheumatic Fever are susceptible to SBE Produces GLUCANS and DEXTRANS promotes attachment to cell surfaces; TOOTH SURFACES Not classified under LANCEFIELD Optochin (Resistant); Bile Insoluble (tests to differentiate from S. pneumoniae); On BAP: alpha-hemolytic Normal flora of the URT, GIT, GUT SPECIES: Streptococcus mitis Streptococcus salivarius some strain are BILE ESCULIN (+) Streptococcus constellatus Streptococcus intermedius Streptococcus sanguis causes SUBACUTE ENDOCARDITIS (SBE) due to dental procedure Streptococcus mutans causes DENTAL CARRIES and DENTAL PLAQUE NUTRIONALLY VARIANT STREPTOCOCCI AKA Pyridoxal Requiring, Satelliting Strep, Cell Wall Deficient Strep Abiotrophia and Granulicatella These are S. adjacens and S. defectivus These are viridans strep needing Vitamin B6 for growth Thiol-requiring streptococci (Ex. Cysteine) called SATELLITING-Strep since they satellite around colonies of S. aureus because they provide the pyridoxal needed (+) Growth in Bile Esculin and 6.5% NaCl Pyridoxal Producing Bacteria Leuconostoc Pediococcus Globicatella S. urinalis Lactococcus S. aureus E. coli Klebsiella spp Enterobacter spp Yeast BACTERIOLOGY GRAM-NEGATIVE COCCI LECTURE Neisseria Non-motile, AEROBIC, capnophilic 5-10%; COLD SENSITIVE; non-hemolytic, most species are CHO fermenters Pathogenic Neisseria are usually fastidious and would usually grow optimally at 35-37 degC Requires enriched media containing IRON, HEMIN, COENZYME which can be found in CAP Most commensal Neisseria can grow on CAP or BAP at room or temp Develops non-pigmented colonies except: N. flava N. flavescens N. subflava All are catalase test (+) except: N. enlongata All are Cytochrome Oxidase Test (+) except: N. elongata Oxidase Test Screening test/presumptive test Reagent: TETRAMETHYL-PPHENYLENEDIAMINE DIHYDROCHLORIDE (+) Result: purple CHO Fermentation Agar: Cysteine Trypticase Agar + 1 Sugar (sucrose, lactose, glucose, fructose) Indicator: Phenol Red (+) Result: Yellow 1. Neisseria gonorrhoeae Kidney, coffee bean shaped diplococci INTRACELLULAR within PMN Ferments glucose only SUPEROXOL TEST A rapid test to detect N. gonorrhoeae Rgt: 30% H2O2 (+) Result: vigorous bubbling Virulence Factor Pili - promotes attachment to host cell Lipopolysaccharide - source of ENDOTOXIN Outer Membrane Protein - affects antibody formation Protein I Protein II IgA Protease Cellular membrane proteins (Por B only) Lipooligosaccharide (LOS) endotoxin Disease causes Gonorrhea “tulo” Opthalmia neonatorum Untreated gonococcal cervicitis may cause sterility and Perihepatitis or FITZ HUGH Curtis SYNDROME Pharyngitis Anorectal Infections (Rectal Gonorrhea) 2. Neisseria meningitidis Bean-shaped diplococci; sensitive to SPS Neufeld Quellung Test (+) Ferments glucose and maltose Natural habitat: ORO and NASOPHARYNX Negative for lactose, sucrose and fructose Serogroups are based on the type of capsular polysaccharide Serogroups include: A, B, C, D, X, Y, Z, 29E, W135 Virulence Factor CAPSULE enable organisms to resists phagocytosis Pili promotes attachment ENDOTOXIN causes petechial formation and DIC IgA PROTEASE important in pathogenesis Cellular Membrane Proteins (Por A and Por B) Lipooligosaccharide (LOS) Endotoxin Disease causes Bacterial Meningitis Meningococcemia organism in the blood; VERY FATAL WATERHOUSE FRIEDRICHSEN SYNDROME severe form of meningococcemia bleeding of adrenal glands and uncontrollable clotting of blood THAYER-MARTIN MODIFIED TM MARTIN LEWIS NY CITY GC-LEC VCN VCN-T VCAn-T VCAm-T VCAm-TL Vancomycin V V V V Colistin C C C C Nystatin Nystatin Anisomycin Amphotericin B Amphotericin B Trimpetophrim lactate Trimpetophrim lactate Trimpetophrim lactate Trimpetophrim lactate Acid produced from spp Glu Mal Lac Dnase N. gonorrhoeae + - - - N. meningitidis + + V - N. lactamica + + + - Moraxella - - - + Moraxella 1. Moraxella catarrhalis Morphologically and biochemically resembles Neisseria Encapsulated with Pili, NON-MOTILE Catalase and OXIDASE (+), BAP - GAMMA Hemolytic A normal flora of ORO and NASOPHARYNX but may cause OTITIS MEDIA It does not degrade sugar (Assacharolytic) BUTYRATE DISK TEST Rapid test to detect M. catarrhalis (+) Result: blue (5-min incubate) Rgt: bromo-chloro-indonyl butyrate DNAse test Best test to differentiate M. catarrhalis to another Moraxella BACTERIOLOGY GRAM-POSITIVE SPORE-FORMING BACILLI LECTURE BACILLUS Spore-forming, rod-shaped organisms; can be isolated from soil They can be AEROBIC or FACULTATIVE ANAEROBES - but only form ENDOSPORES AEROBICALLY It is MOTILE with PERITRICHOUS FLAGELLA except Bacillus anthracis and Bacillus mycoides Biochemical Test: CATALASE (+) and FERMENTS GLUCOSE They can survive in temperature as low as -5 degC and as high as 75 degC due to ENDOSPORES Bacillus cereus Group is the MOST CLINICALLY SIGNIFICANT Species: Bacillus anthracis Bacillus cereus Bacillus thuringiensis Bacillus mycoides 1. Bacillus anthracis HALOPHILIC (7% NaCl) NON-MOTILE forms the so called "DISJOINTED BAMBOO FISHING ROD APPEARANCE" On Gram Staining forms the so called "BOX CAR Morphology" AGENT OF BIOTERRORISM Oval centrally-located spores Lechitinase test (+) Culture media - Egg yolk agar (+) Result Opaque zone around the colonies Produces acid from Glucose, sucrose and maltose Virulence Factor D-glutamate capsule not a polysaccharide unlike the others, MC FADYEAN'S CAPSULAR STAIN and MALACHITE GREEN EXOTOXIN EDEMA factor, LETHAL FACTOR, PROTECTIVE ANTIGEN Theses EXOTOXINS are individually nontoxic but act together to produce damaging effect PROTECTIVE ANTIGEN + LETHAL FACTOR DEATH PROTECTIVE ANTIGEN + EDEMA FACTOR EDEMA Disease causes ANTHRAX A. CUTANEOUS ANTHRAX MOST COMMON; LEAST SEVERE May develop BLACK ESCHAR at the site of entry Black ESCHAR: black necrotic central area or malignant pustule and it is PAINLESS and does not produce pus Organisms enters through cut B. PULMONARY Acquired through inhalation of spores during shearing or sorting of animal hair C. INTESTINAL MOST SEVERE FORM; LEAST COMMON Ingestion of improperly cooked infected meats and ingestion of spores Bloody diarrhea 2. Bacillus cereus Motile and on BAP - B-hemolytic Blood bank contaminant at RT Grayish to lavender color on blood agar Produce acid from glucose, maltose, salicin Lecithinase (+) Virulence Factor Emetic toxin Caused by production of HEAT-STABLE EMETIC TOXIN SHORT INCUBATION (1-6 hours) Vomiting occurs within 6 hours after ingestion of contaminated food Diarrheal Form Caused by production of a HEAT-LABILE ENTEROTOXIN LONG INCUBATION PERIOD (8-16 hours) Toxin is produced when foods are left at RT Disease causes Commonly associated with food poisoning in rice, cereal, vegetables and milk Septicemia, pneumonia, meningitis and peritonitis Eye infections associated with trauma 3. Bacillus subtilis MOTILE and on BAP - Beta Hemolytic Opportunistic pathogen HALOPHILIC (7% NaCl) Gram (+) rod in chain; CENTRAL SPORE Source of Bacitracin (Taxo A) Penicillin - Resistant Selective media: Mannitol egg yolk polymyxin B Agar Common laboratory contaminant Used in Guthrei Test, a microbiologic tes for phenylketonuria Quality control for sterilization CAUSE EYE INFECTION IN Heroin Addict 4. Bacillus stearothermophilus FLAT SOUR SPOILAGE in canned goods Biological Indicator of AUTOCLAVE; no gas but with acid 5.. Bacillus thuringiensis pesticides and insecticides, wound, burn and pulmonary disease An insect pathogen B. anthracis B. cereus Catalase + + Lecithinase + + Motility - + Capsule + - Hemolysis on BAP gamma beta Growth at 45°C - + Salicin fermentation - + Penicillin G S R Gelatin hydrolysis - + PEA - + CLOSTRIDIUM Strict ANAEROBES but some are aerotolerant It is frequently encountered in EXOGENOUS ANAEROBIC INFECTIONS or INTOXICATIONS Catalase negative, Bacillus are Catalase (+) CONTRIBUTE TO VIRULENCE: COLLAGENASE HYALURONIDASE, LECITHINASE PHOSPHOLIPASE Clostrisum sordelii - the only urease (+) Clostria All are motile except: C. perfringens C. ramosum C. septicum All are with swollen spores except: C. perfringens C. septicum All are non-encapsulated except: C. perfringens All are lactose (-) except: C. perfringens C. septicum All are sucrose (-) except C. perfringens All are single hemolytic except C. perfringens All are single hemolytic except C. perfringens All are saccharolytic except C. tetani All are dextrose (+) except C. histolyticum C. tetani Media used Egg Yolk Agar to detect the activities of Lipase and Lecithinase Peptone-Yeast Glucose Broth (PYG) detects VOLATILE FATTY ACIDS CCFA for C. difficile detection only Tranposrt medium: PRAS (Pre-reduced Anaerobically Sterilized) Amies TYPES OF CLOSTRIDIUM NEUROTOXIC HISTOTOXIC ENTERIC C. tetani C. perfringens C. difficile C. botulinum C. septicum C. novyi C. chauvoei C. bifermentans 1. Clostridium perfringens / C. welchii Frankel's Bacillus/Gas Gangrene Bacillus AEROGENIC, Encapsulated, non-motile, double hemolysis Box car shape bacillus Source: wound contact with soil ROUND, SUBTERMINAL SPORES Disease Caused Gas gangrene - myonecrosis Food poisoning - enterotoxins Necrotic enteritis - Pig-bel: a form of bloody diarrhea 2 type of FOOD POISONING: TYPE A: mild and self-limiting GI illness Type C: serious but rarely encountered; bloody diarrhea and vomiting Laboratory Diagnosis Chopped meat agar growth + gas; anaerobic growth Reverse CAMP test KNOWN: S. agalactiae Unknown: C. perfringens (+): arrow head zone of beta hemolysis BAP: Double zone of hemolysis Inside: B-hemolysis (theta toxin) Outside: alpha hemolysis (alpha toxin) Nagler's test/Lecithinase due to alpha toxin, lecithinase, phospholipase C Media: McClung/Neomycin Egg Yolk (+) opalescence on agar w/o antitoxin; inhibition of lecithinase reaction (-) no opalescence on agar w/ antitoxin Stormy fermentation of milk (+) coagulate casein/clotting of protein + gas Litmus Milk test (pH) Acid - pink Alkaline - blue 2. Clostridium tetani IRON LOVING BACTERIA Virulence factor: EXOTOXIN TETANOSPASMIN an exotoxin binds to ganglioside receptors which block release of NEUROTRANSMITTER and inhibits neurons in CNS spastic paralysis TETANOLYSIN cause beta-hemolysis INCUBATION PERIOD: 3-21 days Disease Caused SPASTIC PARALYSIS: with little movement TETANUS: lock jaw "trismus" Risus sardonicus or Sardonic smile Opisthotonus - arching of the back TETANUS NEONATORUM - contaminated instrument used for newborns Laboratory Diagnosis Clinical findings - basis of diseases Morphology test - gram stain TERMINAL OVAL SPORE Tennis racket, drumstick Tack head bacillus Non-saccharolytic 3. Clostridium botulinum VON ERMENGEN'S BACILLUS Canned good bacillus (homemade) NOT CULTURED Virulence: Botulinum toxin which block the release of acetylcholine producing flaccid paralysis Botulinum Toxin, a neurotoxin and the MOST POTENT EXOTOXIN Seven (7) types of Botulism Toxin A, B, C, D, E, F, G A, B, E are associated with HUMAN INFECTION Disease Caused FLACCID PARALYSIS no movement Food botulism home-canned good with preformed toxin Wound botulism spore on wound Wound botulism spore ingestion via breast feeding reduce muscle strength (Hypothonia) FLOPPY BABY SYNDROME SIDS - "SUDDEN INFANT DEATH SYNDROME" aka crib death Laboratory Diagnosis On BAP : beta-hemolysis (+) Lipase Reaction: Eye Yolk Agar Lecithinase (-); Indole (-) Ferments GLUCOSE OVAL, SUBTERMINAL SPORES Diagnosis is done by detecting NEUROTOXIN in serum, feces, gastric contents, vomitus or food MOUSE NEUTRALIZATION TEST DEFINITIVE ID TEST for C. botulinum Detects the presence of NEUROTOXIN 4. Clostridium difficile Colon flora Major cause of diarrhea in hospital OVAL, SUBTERMINAL SPORE Disease Clindamycin: Antibiotic associated with pseudomembranous colitis Found in the patients who have received one or more broad spectrum antibiotics Produces toxin, leading to diarrhea It produces GLUTAMATE DEHYDROGENASE for determination of C. difficile toxin Laboratory Diagnosis Direct detection of toxin from the 81001 by enzyme immunoassay Tissue/Cell culture/Cytotoxin assay GOLD STANDARD for toxin identification of C. difficile Cycloserine Cefoxitin Fructose Agar (CCFA) (+) YELLOW Color (fructose fermentation) Horse-stable or banyard odor indicator: Neutral red BACTERIOLOGY GRAM-POSITIVE NON-SPORE-FORMING BACILLI LECTURE Listeria monocytogenes Gram positive rod or coccobacillus Primarily an ANIMAL PATHOGEN Motile at RT; HALOPHILIC (10% NaCl) TUMBLING MOTILITY on broth at RT (wet mount and hanging drop) PRESUMPTIVE TEST UMBRELLA-LIKE or INVERTED CHRISTMAS TREE MOTILITY on semi-solid on SIM media FACULTATIVE ANAEROBE Listerolysin O (Hemolytic and Cytotoxic) 02 labile hemolysin Allows survival of organism within phagocytes Disease MOT: IN-UTERO/PREGNANCY Neonatal meningitis Sepsis Infanseptica granulomatous Fetal abortion Stillbirth Ingestion of contaminated food (Coleslaw of softcheese) Food poisoning Specimen: Blood, CSF and Tissues Laboratory Diagnosis SAP: Narrow band of beta hemolysis Cold enrichment at 4deg.C Culture on MCBRIDE ANTON TEST ocular virulence test organism inoculated on CONJUNCTUVAL SAC of rabbit (+) PURULENT CONJUNCTIVITIS CAMP test (+) KNOWN: Rhodococcus equi / S. aureus (+) block/rectangular/shovel hemolysis CATALASE (+) and Oxidase (-) Ferments glucose, salicin, and trehalose Hippurate and Bile Esculin OH (+) NEONATAL MENINGITIS S. agalactiae MENINGITIS in children < than 5 YEARS OLD H. influenzae 5-29 years old N. meningitidis MENINGITIS in more than 29 years old S. pneumoniae MENINGITIS in elderly/immunocompromised patients L. monocytogenes Erysipelothrix rhusiopathiae Gram positive rod; non-motile Aerobic or FA and nonhemolytic Not part of the human flora Naturally producing H2S on BAP even without indicator Major reservoir: DOMESTIC SWINE Disease ERYSIPELOID BUTCHER'S CUT, DIAMOND CUT, RED DISEASE a localized skin infection, an occupational hazard for those handling meat and poultry and fish self-limiting infection that resembles streptococcal erysipelas Septicemia Endocarditis individual with valve replacement Laboratory Diagnosis Catalase, Oxidase, Esculin Hydrolysis, VP and Urease and Nitrate (-) H2S (+) Glucose and Lactose Fermenter On GELATIN stab TEST TUBE BRUSH, PIPE CLEANER, BOTTLE BRUSH PATTERN OF GROWTH L. monocytogenes E. rhusiopathiae Beta-hemolysis + alpha or gamma Growth at 4 degC + (-) Catalase Test + (-) Motility + (-) Esculin Hydrolysis + (-) H2S on TSI - (+) VP Test + (-) Gluconate + (-) Media MCBRIDE BAP Lactobacillus aka DODERLEIN BACILLUS Produced large amount of lactic acid Normal flora of mouth, GIT and vaginal canal AEROTOLERANT ANAEROBE Tolerates HIGH ACIDIC ENVIRONMENT (pH of 3.0-4.0) Disease Non-pathogenic and has little clinical significance High during pregnancy Inhibits G. vaginalis, Prevotella, Mobiluncus Promote C.albicans Laboratory Diagnosis Cultivated using TOMATO JUICE AGAR Presence of this bacterium may be noted for PAP'S SMEAR Catalase (-) and NONMOTILE Corynebacterium Majority of the species are found as NORMAL FLORA on SKIN and mucous membrane Closely related to Mycobacteria and Nocardia the cell walls of Corynebacterium contain MESODIAMINOPIMELIC ACID (m-DAP) as the diamino acid and short chain mycolic acid but NON- acid fast Diphteroids normal flora CLUB SHAPE, CHINESE LETTERS, palisade, X and V letters shape All are GLUCOSE and MALTOSE FERMENTERS except C. urealyticum C. pseudodiphtheriticum Laboratory Diagnosis BAP - raised, translucent, gray colonies Catalase and Oxidase positive Babes-Ernst Metachromatic granules NON-motile, NO spore, NO capsule Pleomorphic gram (+) rods Listeria Corynebacterium Motility (+) (-) Esculin HOH (+) (-) Salicin (+) (-) CAMP (+) (-) Corynebacterium diphtheriae aka KLEB-LOEFFER’S BACILLUS It inhabits human nasopharynx but only in carrier state; not part of the normal flora of respiratory tract NONMOTILE, highly pleomorphic (due to irregular snapping) Aerobic (Grows BEST) or FACULTATIVE ANAEROBE It is very resistant to DRYING and remains viable in the environment for weeks One end typically swollen club-shaped appearance; pallisade arrangement -side by side arrangement, picket fence arrangment, X,Y,V, I or CHINESE LETTER FORMATION With BABE-ERNST GRANULES; On BAP it produces narrow zone of beta hemolysis Virulence Factor DIPHTHERIA TOXIN It is produced by strains infected with a lysogenic beta-phages, which carries the tox gene. It inhibits protein synthesis in eukaryotic cell Disease Diphtheria a droplet infection characterized by low grade fever, mild sore throat and body weakness Pseudomembrane in the tonsils, pharynx, larynx due to production of exotoxin, which can eventually cause respiratory obstruction (Bull's Neck or Swollen Neck) Immunization DPT (Diphtheria, Pertussis, Tetanus) Culture Media BLOOD AGAR PLATE have narrow zone of beta-hemolysis LOEFLLER’S SERUM AGAR / PAI’S SLANT Coagulated egg serum will enhance pleomorphism and granule formation (BabeErnst) (+) Poached egg colonies TINSDALE MEDIUM (WITH TELLURITE) It contains sheep's blood, bovine serum, cystine, potassium tellurite and Na thiosulfate (+) Black colonies with brown halo CYSTINE TELLURITE BLOOD AGAR a modification of Tinsdale medium PRIMARY MEDIA FOR ISOLATION; Gray to black colonies It contains sheep's blood, bovine serum, cystine and potassium tellurite (INHIBITOR) (+) Result: BROWN HALO around colonies Toxin Demonstration 1. MODIFIED ELEK TEST IN-VITRO Toxigenicity Test On agar media, place a filter paper strip with diphtheria anti-toxin Streak positive control at right angle to the strip of anti-toxin, streak negative control in the same manner. Unknown culture suspected of C. diphtheriae is streaked parallel to + and - control. Incubate at 35 degC for 24-48hrs. Lines of precipitation/Line of precipitation with ARC IDENTITY is the (+) Result Toxin Demonstration 2. ANIMAL INOCULATION / LETHAL TEST IN-VIVO Toxigenicity Test Suspension of isolated strain of C. diphtheriae (0.3 mL) is injected subcutaneously into thigh of TWO GUINEA PIGS One protected intramuscularly with 500 units of diphtheria antitoxin 18-24 hours before the test (+) Result: THE UNPROTECTED ANIMAL DIES WITHIN 2-3 DAYS with evidence of hemorrhage in the adrenal glands Other Corynebacterium 1. Corynebacterium jeikium "JK BACILLUS"; formerly GROUP JK Resistant to several antibiotics Associated with endocarditis, pneumonia and peritonitis PROSTATIC HEART VALVE Pinpoint white colonies in SBA after incubation at increased CO2 at 30 degC 2. Corynebacterium ulcerans Produces diphtheria-like toxin and diphtheria like infection MASTITIS in cattles, human infections maybe acquired following exposure to infected cattles or consumption of contaminated milk 3. Corynebacterium pseudotuberculosis Formerly known as Corynebacterium ovix, rare cause of granulomatous lymphadenitis An animal pathogen - humans acquired infection through animal contact 4.Corynebacterium xeroxis Normal flora of the skin, nasopharynx and conjunctiva; CONJUNCTIVITIS 5. Corynebacterium pseudodiphthericum "HOFFMAN'S BACILLUS" Normal flora of human oropharynx/oral flora Endocarditis through dental procedure 6.Corynebacterium minutissimum Causes "ERYTHRASMA" - skin infection affecting axillary and pubic skin, produces coral red fluorescence when exposed to wood's lamp due to porphyrin 7.Corynebacterium urealyticum Common isolated in laboratory causes UTI; rapid urease, catalase (+) Laboratory Diagnosis Urease Reduced NO3 test Gelatinase Tellurite C. diphtheriae (-) (+) (-) (+) C. ulcerans (+) (-) (+) RT (+) C. pseudodiphtheriticum (+) (+) (-) (+) C. jeikeium (-) (-) (-) (-) C. pseudotuberculosis (+) V (-) (-) C. urealyticum (+) (-) (-) (-) BACTERIOLOGY ACID-FAST BACILLI LECTURE Mycobacterium Acid fast bacilli due to mycolic acid - acid alcohol resistant Slow growers and requires whole egg for growth except: M. fortuitum M. chelonei With MUCH GRANULES Aerobic, non-sporeformer, non-motile All are aerobic, non-sporeformer except M. marinum 3 GROUPS OF MYCOBACTERIA 1. Mycobacterium tuberculosis complex a. M. tuberculosis b. M. bovis c. M. africanum d. M. canettii e. M. microti 2. Mycobacterium leprae 3. Mycobacteria other than tuberculosis Mycobacterium tuberculosis aka Koch’s Bacillus With "MUCH GRANULES" Size: 1-4um and Diameter (Length): 0.2-0.6um Produces CAULIFLOWER-LIKE GROWTH at 35-37 degC Rough colonies exhibit "CORDING" - colonies appearing like curve strands OBLIGATE AEROBE requires CO2 for growth It is inhibited by NITROIMIDAZOPYRAN It has the LONGEST REPLICATION TIME among the Mycobacteria (20-22 hrs) Types of TB 1. Primary TB initial exposure TUBERCLES: forms granulomatous lesions Caseation Lesions: as the center of tubercles break down, cheese-like masses develop If such lesions calcify, they are termed GHON COMPLEXES, which are observed in a chest x-ray 2. Secondary TB repeat of previous infection 3. MDR-TB (Multi-drug resistant-TB) the type that shows resistance to Primary Drugs (Isoniazid & Rifampin) 4. XDR (Extensively drug resistant) type of TB that shows resistance to Primary TB Drugs, one of the fluoroquinolones, as well as resistant to at least one of the second line injectable TB drugs amikacin, kanamycin or capreomycin 5. POTT’S DISEASE A skeletal TB of the spine 6. Granuloma is an organization of lymphocytes, macrophages, giant cells, fibroblasts and capillaries. TREATMENT PRIMARY Rifampin (Rifampicin) Isoniazid Pyrazinamide Ethambutol Streptomycin SECONDARY Injectables Amikacin Aminoglycosides Capreomycin Kanamycin Fluoroquinolones Ciprofloxacin Ofloxacin Levofloxacin Moxifloxacin SUSCEPTIBILITY TEST 1. Serpentine cord formation 1. MANTOUX TEST Intradermal (forearm) injection (Tuberculin syringe) of PPD PPD (Purified Protein Derivative) is an extract of M. tuberculosis, heat-killed, filtered and precipitated with ammonium sulfate It is used to test if a person has been exposed to TUBERCULIN PROTEIN, either from a PREVIOUS TUBERCULOSIS VACCINATION, or from ENVIRONMENTAL EXPOSURE TO BACTERIA (+) Result: Hard, dense, raised wheal (induration) that is ≥10mm after 48hrs/ If HIV px, >5mm 2. Von Pirquet Test Scratch Test using old tuberculin (OT) + LANOLIN (+) REDNESS 3. VOLLMER PATCH for infants TUBERCULIN TESTS PPD Mycobacterium bovis It causes TB in CATTLES, dogs, cats, swine and parrots It causes Intestinal TB in man COLONY - "Appearing like water droplets" in MIDDLEBROOK Its attenuated strained is used for vaccination BCG (Bacille-Calmette-Guerin) among newborns It is acquired by ingestion of contaminated milk from infected COWS or by exposure to infected animals and their carcasses. Biochemical test: Niacin and Nitrate (-) Mycobacterium canetti It is the smooth stain of Mycobacterium tuberculosis It grows more rapidly than M. tuberculosis The first human isolate was from a cervical lymph node of a somatic child; also isolated from an AIDS patient with mesenteric tuberculosis Biochemical test: (+) Niacin and Nitrate Mycobacterium africanum It is associated with human cases of TB in tropical Africa Mycobacterium microti It has been isolated from TB patients in both immunocompetent and immunocompromised patient. Mycobacterium leprae Causes Hansen's Bacillus or leprosy Forms the so-called CIGAR-POCKET ARRANGEMENT Hydrolyze 3,4-dihydroxyphenylalanine (DOPA) Specimen Smears of tissue juice or tissue, skin snips from eye brows and other sites Mode of transmission INHALATION or CONTACT with INFECTED SKIN Culture DEFINITIVE TEST: NON-CULTURABLE on artificial media but can maintained in the lab using MICE FOOT PADS/FOOT PADS OF ARMADILLO Disease LEPROSY Lepros means "SCALY, SCABBY or ROUGH" A chronic infection of the skin, mucous membrane and peripheral nerves Not considered a highly contagious disease 1. Tuberculoid Leprosy / Paucibacillary Benign form; nonprogressive; recovery is possible because it has no immune defect. Leonine Face It is also associated with delayed-type hypersensitivity reaction to antigens Patients exhibits an effective cell mediated immune response Organisms are extremely rare and may not be detected in skin scrapings or biopsy specimens 2. Lepromatous Leprosy / Multibacillary Malignant form; slowly progressive; life threatening Skin lesions and progressive, symmetric nerve damage Lesions in the mucous membrane may lead to destruction of the cartilaginous septum resulting in nasal and facial deformities Patients DO NOT PRODUCE an effective cell mediated immune response Acid Fast Bacilli are abundant on specimen DIAGNOSIS 1. SKIN TESTS A LEPROMIN TEST skin test that uses killed M. leprae B FERNANDEZ REACTION C MITSUDA REACTION 2. LEPRA CELLS AFB within macrophages 3. BASIS OF DIAGNOSIS Based on symptoms and observation lepra cells 4. WADE FITE FARACO to detect AFB in paraffinized tissues; uses Hematoxylin instead of Methylene Blue as counterstain 5. PHENOLASE TEST separates M. leprae from other mycobacteria with the use of DOPA TREATMENT 1. SULFONE-DAPSONE one of the drugs used for treatment of leprosy LABORATORY DIAGNOSIS OF MYCOBACTERIA SPP. 1. CULTURE GLYCEROL: Carbon source of Mycobacterium Mycobacterium tuberculosis culture are kept in the lab for 6-12 months following identification and susceptibility A NON-SELECTIVE EGG BASED MEDIUM Contains malachite green which inhibits normal flora; growth in 6-10 weeks LOWENSTEIN-JENSEN BEST and the most common egg-based media MTB buff colored, cauliflower non-pigmented colonies AMERICAN THORACIC SOCIETY Less-inhibitory: Sterile specimen like CSF and bone marrow PETRAGNANI for recovery of Mycobacteria from heavily contaminated specimen contains 0.052 g/dL of malachite green Dorset Egg Medium (Wallenstein Medium) Isolation of MAI complex Wallenstein medium for Mycobaterium avium Complex B SERUM-AGAR BASED MEDIA Composed of vitamins, glycerol, malachite green, agar, oleic acid, bovine albumin, glucose and beef catalase Clear media, easy examination of colonies growth in 10-12 days Detection of pigment production Susceptibility testing of MTB Dubol's Oleic Acid Albumin Medium Mitchison's Medium Middlebrook 7H10-7H11 - AST clear media C SELECTIVE With antibiotics contains polymyxin V, amphotericin B, carbenicillin and trimethoprim lactate Gruft Modified Lowenstein Jensen Selective Middlebrook 7H11 Mitchison 7H11 D LIQUID Rapid culture and FASTER TAT These are non-conventional media Bactec 12B (MB 7H12) Specimen volume: 0.5 mL Bactec 13A (MB 7H13) same components with BACTEC 12B + SPS + Polysorbate 80 Specimen Volume: 5mL Septi-Chek AFB Middlebrook 7H9 (broth) rapid culture for MGIT STAINING Mycobacteria possess cell walls that contain MYCOLIC ACID which are LONGCHAIN, multiple cross-linked fatty acid. If at least 2 of the 3 sputum direct smears are (+), then 3 specimens are often sufficient to confirm a diagnosis If none or only one of the first 3 sputum is (+), additional specimens are needed for culture confirmation. 5,000-10,000 AFB/mL - needed for a positive result 1. ACID FAST STAINING It can identify organisms up to Genus level only 2 most common method are Ziehl neelsen - Hot method and Kinyoun's Mehod - cold method AFB will stain RED AGAINST A BLUE BACKGROUND National Standard Reporting Scale - (RIT/DOH) - DSSM DOTS DIRECTLY OBSERVED TREATMENT STRATEGY DSSM DIRECT SPUTUM SMEAR MICROSCOPY SPOT-AM-SPOT 2 sputum (1 morning, 1 random specimen) 2X3 cm ideal size of the smear Dry prior to heat fix to prevent aerosol 70% alcohol with sand - used loop Detection rate of 70% and cure rate of 85% 300 fields examined before negative result REPORT Negative NUMBER OF AFB SEEN (CDC) 0 Doubtful, repeat using new specimen 1-2 AFB/300 fields 1+ 1-9 AFB/ 100 fields 2+ 1-9 AFB/10 fields 3+ 1-9 AFB/fields 4+ >9 AFB/fields FALSE NEGATIVE 1. Overzealous decontamination 2. Loss from concentration techniques 3. Organisms obscured by a too THICK smear 4. Over-decolorizing of the smear 5. Poor counterstaining 6. Lack of observer proficiency in reading stains FALSE POSITIVE 1. Changes in the cell wall 2. Insufficient decolorization 3. Laboratory contamination 4. Delayed processing 5. Overgrowth of other bacteria 2. FLUOROCHROME STAINING PRIMARY STAIN - Auramine rhodamine stain/ TRUANT stain Decolorizer: 5% Acid Alcohol (HCI + Ethanol) Counterstain: 0.5% KMN04 Result: YELLOW AGAINST BLACK BACKGROUND Advantages: EASIER TO READ DUE TO BETTER CONTRAST Auramine is more sensitive than carbol fuchsin AFB are examined at 250x and 400x (LPO) magnification 3. GRAM STAINING To qualify specimen Purpose of digestion and decontamination DIGESTION to liquefy DISULFIDE BONDS in mucus which could trap MTB DECONTAMINATION To remove contaminants as well as normal flora DECONTAMINATION AND DIGESTING AGENTS 1. NaOH (2,3 and 4%) digesting and a decontaminating agent; NOT THE GOLD STANDARD 2. NALC(N-acetyl-L-cysteine) aka DITHIOTREITOL, Sputolysin AND 2-4% NaOH Gold standard NALC- Digestant 2-4% NaOH- Decontaminant 3. Zephiran (Benzalkonium chloride) and Trisodium phosphate/ Z-TSPtyl-L-cysteine) A decontamination-digestion agent Zephiran is an effective decontaminant with little bactericidal effect on the tubercle bacilli TSP (Trisodium phosphate) liquefies sputum rapidly but requires a long exposure time to decontaminate the spx. Phosphate buffer - results in greater isolation of mycobacteria ADVANTAGE: for specimens containing large number of bacteria 4. 1% Cetylpyridium chloride can prolonged shelf-life of sputum for 8 days 5. 5% Oxalic Acid may be added to specimens likely to contaminated by Pseudomonas aeruginosa (cystic fibrosis) Specimens that require decontamination Sputum, Voided urine Autopsy tissue, Abdominal fluid Specimens that require both decontamination and digestion Sputum, Gastric washing, BAL Bronchial washing, Transtracheal aspirate Specimens that do not require decontamination CSF, Synovial fluid and biopsy tissue from deep organs TEST FOR PIGMENTATION This is for the identification of those classified under Runyon's. Mycobacterium Other Than Tuberculosis or Non-TB Mycobacteria Based on: Photoreactivity, Pigment production, Rate of Growth and biochemical test. REPORT 0 NUMBER OF AFB SEEN (CDC) No ABB seem in 300 visual fields +n 1-9/100 fields 1+ 10-99/100 fields 2+ 1-10 AFB/OIF in at at least 50 visual fields 3+ >10 AFB/OIF in at least 20 visual fields GROUP 1 2 GROUP NAME RUNYON’S CLASSIFICATION PHOTOCHROMOGENS Mycobacterium kansasii Mycobacterium asiaticum Mycobacterium marinum Mycobacterium simiae Mycobacterium imtermedium Mycobacterium novocastrene SCOTOCHROMOGENS Mycobacterium scrofulaceum Mycobacterium szulgai Mycobacterium gordonae Mycobacterium thermoresistable Mycobacterium flavescens Mycobacterium cookie Mycobacterium hibernae Mycobacterium heckeshomense Mycobacterium interjecticum Mycobacterium kubicae Mycobacterium tusciae GROUP 3 4 GROUP NAME NONPHOTOCHROMOGENS RAPID GROWERS RUNYON’S CLASSIFICATION Mycobacterium xenopi Mycobacterium ulcerans Mycobacterium haemophilum Mycobacterium gastri Mycobacterium avium Mycobacterium intracellulare Mycobacterium genavense Mycobacterium paratuberculosis Mycobacterium malmoense Mycobacterium fortuitum Mycobacterium chelonei Mycobacterium smegmatis Mycobacterium phlei Mycobacterium abscessus Mycobacterium mucogenicum ADDITIONAL INFORMATION ABOUT NTM 1. Mycobacterium kansasii Also known as the "YELLOW BACILLUS" Tween 80 hydrolysis and Nitrate (+), Rapid catalase activity Causes chronic pulmonary disease 2. Mycobacterium marinum "OF THE SEA", causes skin infections occuring as red to blue lesions and swimming pool granuloma (+) Niacin, Tween 80 Hydrolysis, Urease, Pyrazinamidase (-) Nitrate, Heat-stable catalase 3. Mycobacterium simiae First isolated from MACACA RHESUS MONKEY (+) Niacin, High Heat stable catalase activity, slow hydrolysis of Tween 80 4. Mycobacterium scrofulaceum Smooth, buttery, yellow to orange colonies Causes cervical lymphadenitis (+) Heat Stable Catalase (-) Tween 80 Hydrolysis and Nitrate 5. Mycobacterium asiaticum Similar to M. simiae but negative for niacin 6. Mycobacterium szulgai Photochromogen at 25 degC but scotochromogen 7. Mycobacterium gordonae "TAP WATER BACILLUS"; previously known as M. aquae (+) Tween 80 HOH and Heat -Stable catalase 8. Mycobacterium xenopi Optimally grow at 42 degC Branching colonies with aerial hyphae on cornmeal agar, described to be "bird's nest" (+) Catalase and Arylsulfatase (-) Niacin and Nitrate 9. Mycobacterium avium-intracellulare BATTEY BACILLUS MOST BIOCHEMICAL REACTIONS ARE (-) except for HEAT- STABLE CATALASE and Pyrazinamidase(+) 10. Mycobacterium malmoense (-) Niacin and Nitrate Reduction (+) Tween 80 HOH and Heat-stable catalase (+) 11. Mycobacterium terrae-trivale complex Can cause arthritis and osteomyelitis M. Terrae is also known as the "Raddish Bacillus" 12. Mycobacterium haemophilum Requires hemin for growth 13. Mycobacterium fortuitum-chenolei The only rapid growers that produces a (+) arylsulfatase BIOCHEMICAL TESTS 1. NIACIN TEST All Mycobacterium produce niacin and most possess an enzyme that converts free niacin to niacin ribonucleotide Reagents: strips containing CYANOGEN BROMIDE Organisms is inoculated into the strips (+) RESULT: YELLOW Positive Mycobacteria: M. tuberculosis M. simiae 2. NITRATE Detects the production of NITROREDUCTASE w/c converts NO3 to NO2 Methods: Filter Paper strips (+) Result BLUE COLOR Broth Method Uses SODIUM NITRATE BROTH Indicator reagent: ZINC (detects NITRATE) Reagents: Sulfanilic Acid (HCL sulfanilamide), N-1-naphthylethylenediamine (+) Result: PINK/RED Positive Mycobacteria: M. tuberculosis M. kansasii M. szulgai M. fortuitum 3. CATALASE AND HEAT-STABLE CATALASE Most Mycobacterium are (+) for catalase, but not all of them are (+) for heatstable catalase Heat stable catalase: resistant to heat at 68 degC for 20 minutes Heated at 68 degC for 20 minutes, cooled and reacted with 1mL 30% hydrogen peroxide (+) Result: VIGOROUS BUBBLING Semi-quantitation of catalase production Uses TWEEN 80 and hydrogen peroxide. Reaction is read after 5minutes. The resulting bubbles is measured Results: >45 mm or <45 mm Positive Mycobacteria M. tuberculosis complex 4. IRON-UPTAKE For rapid growers Detects the ability of organism to grow at 20% FERRIC CITRATE and the ability to convert FERRIC AMMONIUM CITRATE (reagent) into IRON OXIDE (+) Result: RUSTY-BROWN COLONIES Positive Mycobacteria M. fortuitum and other rapid growers 5. ARYLSULFATASE For rapid growers Detects the ability of the organism to produce ARYLSULFATASE Arylsulfatase splits PHENOLPHTHALEIN DISULFATE into FREE PHENOLPHTHALEIN, sulfate group and aromatic rings (+) Result: PINK color due to release of phenolphthalein Media: PHENOLPHTHALEIN MEDIA Reagent: SODIUM BICARBONATE + K+ PHENOLPHTHALEIN SULFATE Positive Mycobacteria M. fortuitum M. chelonei 6. TWEEN-80 HYDROLYSIS Detects production of TWEEN 80 LIPASE Tween 80 lipase is an enzyme that splits the detergent TWEEN 80 into oleic acid and polyoxyethylated sorbitol (+) PINK Indicator: PHENOL RED/NEUTRAL RED Positive Mycobacteria M. kansasii 7. PYRAZINAMIDASE TEST Detects production of enzyme PYRAZINAMIDASE Pyrazinamidase splits pyrazinamide into pyrazinoic acid and ammonia Reagent: Ferrous Ammonium Citrate (+) Result: RED PIGMENTATION Positive Mycobacteria M. tuberculosis M. marinum 8. TELLURITE REDUCTION Useful in detection of MAI COMPLEX Ability to reduce colorless potassium tellurite into black metallic tellurium (+) BLACK COLOR Positive Mycobacteria M. tuberculosis M. marinum 9. UREASE TEST Detects production of UREASE A loopful of organisms is placed on urea broth at 37 degC for 3 days Indicator: PHENOL RED (+) PINK Positive Mycobacteria M. scrofulaceum INHIBITORY TESTS 1. NaCl TOLERANCE TEST Uses an egg-based media with 5% NaCl Most Mycobacteria cannot grow in 5% NaCl (+) GROWTH (-) No Growth M. triviale M. flavescens 2. TCH/T2H Susceptibility Test T2H (Thiophene-2-carboxylic acid hydrazide) Distinguish M. tuberculosis (Resistant) from M. bovis (Sensitive) M. tuberculosis (Resistant) 3. Growth on McConkey without Crystal Violet M. fortuitum and M. chelonei complex can grow on MacConkey Agar without crystal violet M. fortuitum & M. chelonei BACTERIOLOGY GRAM NEGATIVE FERMENTATIVE BACILLI LECTURE FAMILY ENTEROBACTERIACEAE Gram negative enteric coccobacilli, short, plump bacilli Non-sporeformers FACULTATIVE ANAEROBES Antigenic: Cell wall (0) somatic, HEAT STABLE, lipopolysaccharide Flagella (H) flagellar, heat labile, protein Capsule (K) capsular, heat labile, polysaccharide K1 - E. coli Vi - S. typhi BAP/CAP: large moist gray colonies except Klebsiella and Enterobacter All are gamma hemolytic except. All are non-encapsulated except. All are GLUCOSE FERMENTERS and often with gas production aerogenic except Shigella All are motile (peritrichous) at 37'C except SKY Shigella, Klebsiella and Yersinia pestis All Yersinia are motile except Y. pestis at 25'C All are CATALASE POSITIVE except All are CYTOCHROME OXIDASE NEGATIVE except All are NITRATE REDUCERS except Most are commensal flora of the intestinal tract except Salmonella, Shigella and Yersinia Some are with pili or fimbrae; Grows well on McConkey Agar Drug of choice: Aminoglycosides, Trimethoprim-SXT, 3rd Gen Cephalosporins VIRULENCE FACTORS 1. Pili used for attachment colonize the area and invade tissues 2. PLASMID resistance to antibiotics 3. ANTIGENS may be used to identify other groups ANTIGENIC STRUCTURE 1. _______________ For E. coli there are 164 types of O antigen and specific type maybe associated with a particular disease HEAT STABLE; LIPOPOLYSACCHARIDE SEROTYPE 0111 - Diarrhea in INFANTS SEROTYPE 0157 - Verotoxin Production For E. coli and Shigella serotyping 2. _______________ Consists of capsular POLYSACCHARIDE HEAT LABILE K1 antigen - E. coli Vi Antigen - S. typhi 3. _______________ Protein in nature HEAT LABILE Found only among motile enteric For Salmonella serotyping MEDIA USED FOR FAMILY ENTEROBACTERIACEAE MEDIA INHIBITORY CHO INDICATOR Eosin Y and Methylene Blue COLONY COLOR Fermenter Non-Fermenter Lactose Eosin Y and Methylene Blue Pinkpurple colonies Colorless Crystal Violet and Bile Salts Lactose Neutral Red Pink Colorless Bile salts Xylose, Lactose, Sucrose Phenol red Yellow Red/Colorless Bile salts Salicin Lactose Sucrose Bromthymol blue yellow Green/Colorless Bile Salts Brilliant Green Lactose Neutral Red Red Colorless MEDIA INHIBITORY COLONY COLOR CHO INDICATOR Brilliant Green GLUCOSE Bismuth Sulfite Bile Salt pH = 8.6 Thymol Blue SUCROSE and Bromthymol blue Fermenter Non-Fermenter SALMONELLA: BLACK Yellow Green/Colorless MEDIA USED FOR FAMILY ENTEROBACTERIACEAE GN Broth For Salmonella Selenite Broth enrichment broth for Salmonella and Shigella Tetrathionate broth enrichment broth for Salmonella CIN (Cefsulodin Irgasan Novobiocin) for Yersinia Brilliant Green Agar (BGA) for other Salmonella except S. typhi Brilliant Green Agar (BGA) for other Salmonella except S. typhi