CLS_A_Bottomline_Approach_5e MICROBIO.pdf

137 MICROBIOLOGY/ Bacteriology by Mona Bakeer and Mary Lux Bacteria...

137 MICROBIOLOGY/ Bacteriology by Mona Bakeer and Mary Lux Bacterial Growth Requirements ❖ Palladium pellets catalyze the 1. T emperature reaction a. P sychrophiles - cold loving; ❖ Some require hemin, Vitamin K , optimum temper ature = 15°C and yeast extract b. Mesophiles - moderate temper ature; 5. Typical incub ation optimum temperature = 37 C; (most a. 5-10% CO2 (incubator or candle pathogenic organism s) j ar ) c. Thermophiles - heat loving; b. 35-37°C optimum temperature = 50-60°C c. 50-70% humidity 2. pH optimized for most 6. May be selective and/or differential a. Bacteria 6.5-7.5 b. Fungi 5.0-6.0 STERIUZATION/INHIBITION TECHNIQUES 1. H eat - denatures p rotein 3. Oxygen a. Moist - autoclave (steam under a. Aerobes - require 0 2 pressure) b. Facultative anaerobes - can grow ❖ 15 lbs pressure/sq. in., 121 °C, with or without 0 2 15 minutes c. Obligate anaerobes - harmed by 0 2 ❖ QC - Bacillus stearothermopbilus d. F ormation of superoxide radical!,, b. Dry heat toxic; n eutr alized by catalase, ❖ Flame, incinerator peroxidase and ❖ Hot air oven 170°C, 2 hrs superoxide dismutase (p ossessed b y c. Pasteuriza tion , ultra high aerobes and fa cultatives) temperature ❖ 140°G, 3 secon ds 4. Other atmosph eric requirements ❖ NOT sterilization a. Microaerophiles - p refer lower 0 2 than :in air 2. Filtration b. Capnophilcs - prefer higher CO2 a. Pore size 0. 22 µ - 0.45 µ than in air b. Used for sugar solutions, urea c. Aerotolerant- do not require 0 2 but media , vaccines not poisoned by 0 2 3. Refriger ation - slows gr owth CULTURE MEDIA 4. Dessication - no multiplication, but 1. Must meet growth r equirements or ganisms r emain viable (Jyophilization) 2. Agar - polysaccharide der ived from marine algae 5. Osmotic pressure hypertonic solution a. Melts at l00°C a. Causes plasmolysis b. Solidifies at approximately 45°C h. "Cured " meat, fruit preserves 3. Complex media - most common ; ma de 6. Radiation of peptones and extracts a. Forms h ydroxyl radicals b. Damages D A 4. Anaerobic media - contain r educing agents which bind with dissolved 0 2 7. Disinfection (tbi.oglycollate, cysteine) a. Phenol - damages cytoplasmic a. Bro th tubes should h e heated prior membrane, denatures protein to use to drive out 0 2 b. Halogen s (iodine and chlorine) - b. Gas pak en velop es oxidizer s ❖ Contain Na2 CO3 and sodium c. Alcohols - denature protein and horohydride dissolve lipids ❖ Add water - produces H2 and CO2 (aids in growth) 138 STERILIZATION 0ISINFErTION Kills All Microorganisms Inactivation or Inhibition of (Including Spores and Microorllanisms (May Not Viruses) A ect Spores) Examples: [xample: Autoclave (121 'C at 1Spsi Bleach (1:10 Hypochlorite) Autoclave Pressure, for 15 min.) Temperature and Time Incineration Filtration (Physically Removes Microorganisms) Antibiotics and Their Actions ANTIBIOTIC EXAMPLES ACTION NOTES B-lactams Penicillins Inhibits cell wall synthesis Ceftriaxone, Cefotax ime Cephalosporins Carbapenams (lmipenam) Monobactams (Azotreonam) B-lactamase Inhibiting Combin- ations (Augmentin, etc.) Glycopeptides Vancomycin Inhibits cell wall synthesis Drug of choice for Clostridium d ifficile and MRSA Aminoglycosides Gentamicin Inhibits protein synthesis Acts on 30S subunit; not active Tobramycin against anaerobes; used with a Amikacin penicillin for Enterococcus Tetracyclines Tetracycline Inhibits protein synthesis Acts on 30S subunit; affects Doxycycline bone an d teeth in children ; may lead to super infection of yeast Chloramphenicol Chloramphenicol Inhibits protein synthesis Acts on 50S subunit; can cause aplastic anemia MacroIides Erythromycin Inhibits protein synthesis Acts o n 50S subunit; Clindamycin clindamycin for GP and G N anaerobes Quinolines Ciprofloxacin Inhibits nucleic acid Fo r Pseudomonas aeruginosa Norfloxacin synthesis and other aerobes Sulfa Drugs (Sulfonomides) Sulfamethoxazole Analogue of PABA For UTI. enteric infections; used (intermediate in folic acid with trimethoprim (Bactrim, etc.) synthesis) Streptogramins Quinupristin/dalfopristin Inhibits protein sythesis For GP, especially vancomycin resistant Enterococcus faecium Oxazdidinones Linezolid Inhibits protein sythesis For GP, including those resistant to other antibiotics Antibiotics/Susceptibi/i'ty Testing 4. Bactericidal action - kills bacteria without host immune help ANTIMICROBIAL THERAPY 1. Narrow spectrum - only certain 5. Bacteriostatic action - rever sible groups covered inhibition ( ultimate destruction d epends on h ost defen ses) 2. Broad spectrum - Gram pos (GP) and Gram n eg ( GN) coverage 6. Drug combination a. Synergism - combined b etter than 3. Selective toxicity - action against the sum: 1 + 2 = 4 microbe only without injuring cells of b. Antagonism - one d ecreases activity host of other: 1 + 2 = l  139 SUSCEPTIBILITY TESTING c. Inducible strains form a ""D" - 1. Kirby-Bauer Method shaped zone of inhibition a. Disk diffusion b. Mueller-Hinton agar (MH) 7. Detection of MRSA c. Depth = 4mm a. Zone of ::;10 mm with an oxacillin d. pH = 7.2-7.4 (1 µg) disk on Mueller -Hinton e. Physiologic concentration of Ca++ b. Molecular tests for mecA gene and Mg++ f. 35°C, ambient air 8. Vancomycin-Resistant Enter ococci g. 108 organisms (McFarland 0.5) (VRE) h. QC weekly and with each n ew lot of 9. Carbapenem r esistant agar or discs (E. coli, S. aureus, enterobacteriaceae (CRE) P. aeruginosa) a. Enzymes resistant to Ca rhapenem antibiotics 2. Broth methods a. MIC (minimum inhibitory concentration) b. Klebsiella spp. especially K. pneumonia (KPC). E. coli, ❖ Lowest concentration of drug that Enter obac ter prevents in vitro growth ❖ First dilution tube with no visible c. Resistant to penicillin, cephalosporins, growth carbapenem and aztreonam b. MBC ( minimum b acteriostatic d. Modified Hodge test and chromogenic media concentration) ❖ Lowest cor1centration that results 10. Automated AST in >99.9% killing a. BD Phoenix ❖ Subculture l'Ubes near MIC to find b. Microscan Walkaway first plate with no growth c. TREK sensititre 3. E-test d. Vitek l and 2 a. MIC on a stick Sources of Error: Disk Diffusion b. Plastic strips impregnated with ABNORMAi RE'" 'LT PROBARI F rAI 1,~ antimicrobials Tetracycl ine Zone Too Large pH of agar too low and Clinda17.cin Too 4. Modified methods for testing slow- Small with. coli or growing or fastidious bacteria S. aureus Controls a. Haemophilus test medium Tetracycline Zone Too Small pH of agar too high Jnd ClindJmycin Too b. Supplemented MH for S. Large with E. coli or S. aureus Controls pneumoniae c. Supplemented GC agar base for N. Amino~lycoside Zone Too Ca++ and/or Mg++ Sma with P. aeruginosa too high in agar gonorrhoeae Control Aminoglycoside Zone Too Ca++ and/or Mg++ 5. Extended spectrum heta-lactamase large with P. aeruginosa too low in agar Control (ESBL) Zones Universal!)' Too La rgc lnoculum too light a. Enzymes for resistance to extended- on Control Plates Nutritionally poor medium spectrum (third-generation) of Slow.growing organism (not seen with controls) cephalosporins and monobactams Agar depth too thin but do not affect cephamycins Zones Universally Too Small lnoculum too heavy b. Enzyme activity may vary on Control Plates Agar depth too thick c. If an ESBL is detected, all Methicillin Zone Dccreasin~ Methicillin degrading during over Days or Weeks wit refrigerator storage penicillins, cephalosporins, and Control Or~anisms aztreonam should be r eported as Methicillin Zone Methicillin being degraded resistant Indeterminate in Disk Test by strong &--lactamase producing Staphylococci d. E specially consider E scherichia and Colonies within Zone of Mixed culture Klehsi ella as potential ESBL- Inhibition Resistant mutants within producing organisms zone "Zone within a Zone" A swarming Proteus Phenomenon Feather edges of zones 6. D-test around ~enicillin or a. Used for detection of inducible ampicil in disks usually occur with B lactmase clindamycin r esistance. neg. strains of 5. aureus b. Clindamycin 2µg disks and B lactamase pos. H. influenzae with erythromycin 15 µg disks used penicillin or ampicillin 140 Media Routine Media Special Media MEDIA PURPOSE MEDIA PllRPO'-f Blood A~ar Most Bacteria; Determines Bordet-Gengou Agar 8. pertussis (BA, BA ) Hemolytic Reactions Buffered Charcoal Legionella sp. Chocolate Agar Haemophilus and Neisseria sp.; Yeast Extract (BCYE) Enriched with Hemoglobin or lsoVitalleX Cystine-Glucose Agar F. tularensis Phenylethyl Alcohol Selects for Gram Positive Cocci and Fletcher's Medium leptospira Agar (PEA) Anaerobic Gram Negative Bacilli Skirrow Agar Helicobacter pylori Columbia Colistin- Selects for Gram Positive Cocci Nalidixic Acid (CNA) Thiosulfate Citrate-Bile Vibrio sp. Salts Sucrose (TCBS) Vaginal is Agar (V-Agar) Cardnere/la vagina/is Thayer-Martin Agar N. gonorrhoeae and N meningitidis (human blood) CAMPY-Blood Agar Campylobacter sp. Cystine-Tellurite Blood C. diphtheriae (Black Colonies) Thioglycolate Broth "Back-Up" for Anaerobes (Tinsdale) Lowenstein-Jensen Mycobacterium sp. Loeffler's Medium C. diphtheriae (Enhances Grouping Agar and Metachromatic Granules) Middlebrook 7H10 Mycobacterium sp. Agar Select Biochemical Reactions Petragnani Agar Mycobacterium sp. Biochemical Test Uni nocu lated Positive Medium Color or Negative Reaction Reaction Anaerobic Media Carbohydrate Red Yellow MEDIA PURPOSE ferrnenatation with phenol red Bacteroides Bile Selects for B. fragilis Group Esculin Agar (BBE) (Black Colonies) Esculin Nondescript Black Kanamycin- Bacteroides sp. Hippurate No change Purple Vancomd1cin Laked (Enhances Pigment Production) hydrolysis Bloo (KVLB) Motility Growth along Blurred stab Cycloserine-Cefoxitin C. difficile stab line line Fructose Agar (CCFA) 6.5% NaCl Broth Clear Turbid CDC Anaerobic Blood Anaerobes (Enriched with Hemin, Phenylalanine or Nondescript Green Agar Cystine, and Vitamin K) trytophan surface after Cooked or Chopped Anaerobes deaminase 10% FeCl3 Meat Medium Urea Nondescript Bright pink COLD EIIRiCHIIENf Listeria monocytogenes Yersinia Anaerobic Media and Associated Organisms Special Media and Associated Organisms Especially Legionella  141 Specimen Collection and Handling GENERAL 1. Material from infection site CoHecti.on and Handling of 2. Optimal time (ex.: Salmonella typhi- Blood Culture Specimem1 culture blood ffrst week, culture stool second and third weeks) Diagnostic Methods 3. Appropriate collection devices MICROSCOPY 4. NEVER refrigerate spinal fluids, 1. Light microscopy anaerobic or GC specimens a. Resolving power - 0.2 µm b. Ocular lens= l0X; oil immersion 5. Collect prior to antibiotic therapy lens= IO0X 6. Set up within 2 hours of collection 2. Darkfield - for spirochetes; reflected CRITERIA FOR REJECTION light 1. Preservatives used 3. Fluorescence - near UV range; auramine rhodamine, acridine orange 2. Insufficient quantity and calco-fluor white stains 3. Dry swab 4. Electron microscopy - can resolve 4. Leaky containers - contaminated particles 0.001 µm apart; u seful in viral specimen as well as biohazard I.D. BLOOD CULTURE COLLECTION CULTURE CONDITIONS 1. Must prep skin properly with alcohol 1. Most plates incubated at 35-37°C (70%) and iodine a. Campylobacter - 42°C b. Yersinia - 25-30°C 2. Best time to draw is just prior to fever c. 5-10% CO2 (Campy- spike mi.croaeropl1ilic) d. 50-70% humidity 3. Draw at least 2 cultures, but no more than 3 in a 24 hr period 2. Anaerobic Conditions a. Broths with thioglyco.llate or 4. May use antibiotic removal device (ARD) cysteine or resin bottles if patient on antil1iotics b. P re-reduced media ❖ Gas pak jars or anaerobic 5. Isolator® best for fungi and acid fast chamber organisms ❖ Environment: 10% H2, 5% CO2, 6. Need 1: 10 dilution of blood to broth; 85% N2, palladium crystals on adults draw at least 10 ml (5ml for MEDIA pediatric bottles) 1. Approximately 1.5% agar 2. Non-selective a. Supports most organism s b. Blood agar, chocolate agar, trypticase soy agar 3. Selective agar a. Contains chemicals, dyes, antibiotics to inhibit certain organisms (EMB, MAC, CNA, Campy-blood) b. May also be differential (HE, SS, XLD, EMB, MAC) 142 INOCULATION 3. Number of colonies x 100 (.OJ loop) or 1. Streak for isolation wit h nichrome or number of colonies x 1000 (.001 loop) pl atinum, or disposable loops 4. Read and report after 18-24 hrs 2. Calibrated 0.01 ml or 0.001 ml (. 001 ml for urine colony count plates) Stains Commonly Used in Microbio/ogy/Mycobacteriology PRIMARY DECOLORIZER COUNTERSTAIN RESULTS PRINCIPLE STAIN POS NEG Gram Stain Crystal Violet Alcohol/Acetone Safranin Purple Pink Iodine mordant Methanol or heat tix Violet dye & iodine form complex in cell; washes out of gram neg cells Kinyoun & Carbol Fuchsin Acid Alcohol Methylene Blue Pink Blue Acid tast Ziehl-Nielson For Mycobacteria Auramine- Auramine and Acid Alcohol Potassium Orange No For Mycobacteria Rhodamine Rhodamine Permanganate Fluoresc. Fluoresc. (Fluorescent Stain) Calcofluor Calcofluor White Bluish- No For yeast and fungi White + 10%KOH white Fluoresc. KOH to break down Fluoresc. debris and mucous Gram Positive Cocci STAPHYLOCOCCUS if methicillin-resistant S. aureus 1. "Grape-like" clusters (MRSA), vancomycin is drug of choice 2. S. a:ureus g. Laboratory diagnosis a. Coagulase positive ❖ BAP - soft, opaque, regular b. Most common pathogen of genus colonies 2-3 mm in diam; some are c. Common infections beta hemolytic and some have pale ❖ Furnncles (boils) and carbuncles golden color ❖ Biillous impetigo (blisters) ❖ Growth in 7.5% NaCl and ferment ❖ Paronycbia (nails) mannitol ❖ Post surgical wounds and ❖ Catalase positive and coagulase bacteremia positive d. Intoxications ❖ Pulse Field Gel Electrophoresis ❖ Scalded skin syndrome (PFGE) and susceptibility (exfoliatin - neonates) proflle for epidemiologi.c studies ❖ Toxic shock syndrome (TSST-1) - women ages 12-52 3. Coagulase negative Staphylococcus ❖ Food poisoning (en terotox.in) - a. Opportunist in symptoms in 1-5 hrs after immunocompromised hosts and ingestion (potato salad , cream patients with prosthetic valves and dislies) devices e. Exotoxins - h emolysins, leukocidins, coagulase and hyaluronidase (spreading factor), nuclease, protease and lipase f. Resistance/sensitivity ❖ Most resistant to penicillin due to Susceptibility Teating of plasmid mediated B-lactamase Staphylococcw ❖ Some sensitive to penicillinase- resistant penicillins (PRP's) (methicillin , oxacillin, etc);  143 Biochemical Tests COAGULASE TEST CATALASE TEST 1. Rea gent - EDTA rabbit plasma 1. Reagent: 3% H202 2. Bound coagulase - clumping on slide 2. Add one drop to colony on slide (plasma and colony) 3. Free coagulase - gels in tube test (0.5 ml 3. If catalase present, H 20 2 is broken plasma and colony; 35-37°C 4-12 hrs) down to water and 0 2 (which bubbles off) 4. Agglutination tests - detect coagulase and protein A 4. Positive: Staph; negative - Strep 5. Positive: S. aureus; negative: other 5. QC each day of use Staph (human pathogens) Staphylococcus (Catalase Positive) ORGANISM COAGULASE INFECTIONS INTOXICATIONS NOTES S. aureus + Carbuncles, furuncles, Scalded Skin Syndrome, Most beta lactamase +; paronychia, wounds, Toxic Shock Syndrome, many MRSA and bacteremia and Gastritis (enterotoxin, CA= community acquired 1-5 hours after eating) HA=hosptial aquired S. epidermidis - Endocarditis, prosthe- Most methicillin resistant; tic device infections sensitive to novobiocin S. saprophyticus - UTI in young women Resistant to novobiocin Coag Negative Staph: Opportunistic Infections in lmmunocompromised Patients and Those with Prosthetic Devices Staphylococcus aureus Staph aureus is my nickname Preparation of Kirby-Bauer In the Plates 1UJd bJoculum pathogen Hall of Fame Look for bubbles Organisms Used for QC in with catalase BUBCeptibility resting And for clots with coagulase. STREPTOCOCCUS Gram positive clusters everywhere Skin and gut, nose and hair. 1. Spherical or oval; chains or pairs Protein A is my main trait 2. Lancefield grouping based on C carbohydrate and Enterotoxin in what you ate. 3. S. p yogenes (Group A) Penicillin won't touch me a. Beta hemolytic Methicillin, well... maybe? b. Streptolysin S - stable in 0 2 ; non- Vancomycin killed in days of yore antigenic But now that drug is not for sure. c. Streptolysin 0 - oxygen labile; Wash your hands, don't pick your nose. antigenic d. Erythrogenic toxin - rash of scarlet Wear a lab coat over your clothes. fever 144 e. Highly sensitive to penicillin f. Infections ❖ Pharyngitis (Strep throat) ❖ Impetigo ❖ Erysipelas ❖ Wounds, burns ❖ Rheumatic fever (a utoimmune compound sequelae to infection with (etbylbydrocupreine Streptococcus g1·oup A) ❖ Lab diagnosis ~ Sensitive to 0.04 units Streptococcus bacitracin disc pyogenes I&' Typing Streptococcus 4. S. agalactiae (Group B) pyogenes a. arrow zone of beta hemolysis Penicillin will kill h. Neonatal sepsis and meningitis; with eose. UTI; vaginal infections c. Laboratory diagnosis But Protein M is ❖ Serotyping the biggest trick ❖ CAMP reaction (with S. aureus) To make throat ❖ Na hippurate positive and muscle sick. 5. Group D Antistreptolysin O a. S. bovis/ gallolyticus Will lay the heart and kidneys low. ❖ BEM positive Bacitracin is my sign ❖ No growth in 6.5% NaCl Zone of inhibition every time. ❖ Associated with colorectal cancer b. Enterococcus See what I do on 8-A-P ❖ Bile esculin medium (BEM) Destroy the sheep R-8-C. positive ❖ Growth in 6.5% NaCl ❖ UTI, bacteremia, others REMEMBER! 6. S. pneumoniae Staph and Strep a. Alpha hemolytic crater -like colonies STREP: or mucoid, "water drop" colonies Pairs or Chains b. Lancet-shaped diplococci Catalase - c. Check sensitivity to penicillin using Growth in 6.5% NaCl OX (oxacillin) disc(~ 20 mm= (Enterococcus) sensitive) d. Cau ses ❖ Primary lobar pneumonia (rusty sputum) Catalase neg ❖ Menin.gitis ❖ Bacteremia STAPH: ❖ Otitis media Clusters (Grape-Like) ❖ Conjunctivitis Catalase + e. Laboratory diagnosis Growth in 7.5% NaCl ❖ Typical colony mo1phology ❖ Quellung reaction ❖ Sensitive to optochin ❖ Bile soluble Catalase pos 7. Other alpha Streptococcus ( viridans group) - subacute bacterial Coagulase + endocarditis (SBE) (S. aureus) (See Streptococci Chart on next page.)  streptococci (Catalase Negative) Hemolysis Bacitracin Na Optochin Bile Bile Esculin 6.5% NaCl Infections Hippurate Solubility S.pyogenes B s R Pharyngitis, wounds, scarlet (group A) fever, impetigo, sequalae-rheumatic fever S. agalactiae B, y R + R Neon atal septicemia and (group B) meningitis, UTI (CAMP+) Entercoccus a, B, y R R + + UTI, endocarditis (treat with (group D) aminoglycoside+penicillin) Non-enterococcus a ,y R R + Endocarditis (rare), Strep. bovis (group D) associated with color cancer Pneumonia, meningitis, S. pneumoniae a ("water drop'1/ R s + bacteremia (screen for crater colonies) penicillin sensitive with Ox; > 20 mm= sensitive) S. viridans a,y R R Endocarditis (rare) I ~ -, er-t: ! a hemolysis = greening ar?und colony o hemolys1s = no hemolys1s B hernolysis = complete clearing around colony Shaded areas = Key Reactions (3 Growth Requirements 3r Strepiococci ~ REMEMBER! ~ Victory Belongs to Key Cbara(iteristics oi the Most Persevering. Pneumococcl.lB...... ~ 146 Gram Negative Cocci 4. Bacterial meningitis- CSF a..+neutrophils NEISSERIA AND MORAXELJ.A b. t" glucose 1. Key characteristics c..+protein a. Diplococci (kidney bean shape) b. Oxidase positive 5. Moraxella (Branhamella) catarrhalis a. Respiratory infections 2. N. gonorrhoeae b. Grows well on chocolate and BAP a. Grows on chocolate and Thayer- but n ot on MacConkey's Martin (contains vancomycin, c. Colony hard and "moves over"; colistin, nystatin, hemoglobin and asaccharolytic isovitalex) d. Usually beta lactamase positive b. Requires 5-10% CO2; may take 48 e. DNAse positive hrs for growth c. Ferments glucose 6. Acinetobacter species d. Gonorrhea a. Emerging pathogen ❖ Sexually transmitted b. Respiratory infection s, UTI , or ❖ May be asymptomatic; may he colonizer mixed with Chlamydia c. Coccobacillus ❖ Gram stain sensitive for males, d. Resistance to many drugs; AST but NOT for females required ❖ May be confused with Moraxella e. Identification or Acinetobacter ❖ Growth on MAC ❖ Do NOT refrigerate prior to ❖ Some h emolytic culture ❖ Oxidase negative ❖ Penicillin or spectinomycin sensitivity; perform bet·a lactamase test to determine penicillin sensitivity @Diffe~~~~~seria e. Most ID by probe technology Glucose Maltose Lactose Neisseria gonorrhoeae N. gonorrhoeae + Gonorrhea, the clap, GC N. meningitidis + + I'm an old-fashioned STD. + N. lactamica + + I need co2 and chocolate All ferment glucose; g_onorrhoeae: only glucose; agar meningitidis: glucose and maltose; and And may not grow for 48 factamica: glucose, maltose and lactose. hours. Gram Positive Rods I ferment glucose, the only sugar for me, CORYNEBACTERIUM DIPH1HERIAE Do an oxidase and purple you'll see. 1. Key Characteristics a. Small pleomorphic rods with 3. N. meningitidis clubbed ends a. Grows on blood agar , chocolate and b. Palisade or "chinese letter" Thayer-Martin arrangement b. 5-10% CO2 enhances growth c. Metachromatic granules (stain c. Ferments glucose and maltose red-purple wit/1 methylene blue) d. Transmitted by respiratory droplets d. Tinsdale agar - black colonies due and requires close contact to tellurite hydrolysis e. Meningitis e. E lek test - determines toxin ❖ Seen mostly in children under 3 production by the isolate in vitro ❖ Waterhouse - Friderichsen syndrome (sca ttered petechiae) = 2. Loeffler' s - enhances development of meningiococcemia m etachromatic granules ❖ Ma.inly caused by types A, B, C, Y a. Pallisade arrangement may be seen &W  147 3. Exotoxin production b y only lysogenic ERYSIPELOTHRIX organsims carrying a B phage 1. Key Characteristics a. Non-motile 4. Produces pseudomembrane on tonsils, b. Catalase negative uvula or soft palate c. "Test tube brush" growth in gelatin; 5. Causes diphtheria H2S positive in TSI GARDNERELLA VAGINALIS 2. Occupational infection for fishermen, 1. Bacte1·ial vaginosis butchers, veterinarians, rose grower s BACILLUS SP. (Sporeformers) 2. "Clue cells" - high number of squamous epithelial cells colonized with 1. Key Characteristics gram variable rods a. Large, ground glass colonies b. Some beta hemolysis (But not B. 3. 10% KOH added to discharge causes anthracis) "fishy" odor c. Catalase positive d. Large gram positive to variable rods 4. Tiny colonies at 48 hrs on BAP and in chains with spores chocolate 2. B. anthracis 5. Catalase and oxidase negative; a. Very long chains ("bamboo shoots'') hippurate and starch positive b. "Medusa h ead" colonies LISTERIA MONOCYTOGENES c. Non-motile, non-hemolytic 1. Key Characteristics d. Anthrax - cutaneous, pulmonary, or a. Small colonies with narrow zone of gastrointestinal beta hemolysis 3. B. cereus - Food poisoning due to b. Catalase positive preformed toxin - "fried rice" c. Tumbling motility; "'umbrella" motility in SIM at r oom temperature but NOT 37°C d. Bile esculin positive 2. Causes neonatal meningitis and sepsis; sepsis in immunocompromised hosts Gram Positive Rods ORGANISM CATALASE ESCULIN H2S /TSI ~ HEMOLYSIS NOTES Corynebacterium + - - +/- "Chinese Letter" Arrangement; r'1) ~ Metachromatic Granules (Loeffler's Slants); Tellurite Hydrolysis ti ~ (Tinsdale Agar); Elek Test Determines Toxin Production Listeria + + - + Tumbling Motility at 250 C, but Not ~ 37C; Cold Enrichment; Neonatal Meningitis and Sepsis; Sepsis in lmmunocompromised Hosts El}',~sipelothrix - - + - 'Test Tube Brush" Growth in Gelatin; Infection in Fishermen, , , Butchers, Veterinarians Bacillus (Spore Formers) + V - - I+ "Ground Glass" Hemolytic Colonies; l)il ~ a D ti rn B. anthracis -Non-Hemolytic, Non-Motile, "Medusa-head" colonies (filamentous outgrowth) B. cereus- betahemolytic, motile , large flat colonies (irregular edges) 148 Gram Negative Rods ❖ Food poisoning associated with ENTEROBACTERIACEAE undercooked meat (hamburger) ❖ Hemolytic uremic syndrome 1. General Characteristics (HUS) a. Peritrichous flagella when motile b. Ferment glucose 4. Kl strains can cause neonatal meningitis c. Reduce NO3 to NOz d. Most oxidase negative Shigella e. Antigens used in typing: 1. Key Ch aracteristics ❖ Flagella = H Ag a. Lactose negative ❖ Envelope = K Ag b. Non-motile ❖ Cell wall LPS c. Anaerogenic (lipopolysaccharide) = 0 Ag ❖ All possess LPS endotoxin; some 2. Bacillary dysentery - penetrate produce exotoxins epithelial cells in small intestine Escherichia coli 3. S. dysenteriae (Group A)- most severe 1. Key Characteristics 4. S. flexneri (Group B) a. Indole and lactose positive b. IMViC = ++ - - 5. S. hoydii (Group C) 2. Most common cause of UTI in females 6. S. sonnei (Group DJ- most common 3. Intestinal infections 7. < 200 organisms needed for disease a. Enterotoxigenic E. coli (ETEC) ❖ LT toxin (heat labile) ❖ ST toxin (heat stable) b Enteroinvasive E. coli (EIEC) ❖ Penetrate epithelial cells in large REMEMBER! intestine Shigella ❖ May be lactose negative c. Enterohem or r hagic E. coli (EHEC) S. dysenteriae = Group A (1st alphabetically) - strain 0157:H7 S. flexneri = Group B (not "B"oydii) ❖ Sbigella-like toxin S. boydii = Group C ("boyd ee ee" = "C'J S. sonnei = Group D (last alphabetically) Common Gram Negative Selective Media AGAR DIFFERENTIATING SELECTIVE H2S LACTOSE LACTOSE AGENT AGENT INDICATOR POSITIVE NEGATIVE MAC Lactose Crystal Violet, None Dark Pink Transparent MacConkey Bile Salts EMB Sucrose Eosin Y None Green Sheen, Transparent (Eosin, Methylene Blue) Lactose Methylene Blue Purplish and Brownish Amber HE Lactose Bile Salts Sodium Thiosulfate Salmon Green to Blue (Hektoen-Enteric) Sucrose (H:zS+ = Black) Salicin ss Lactose Brilliant Green Sodium Thiosulfate Red Transparent (Salmonella - Shigella) Bile Salts (H:zS+ = Black Center) XLD (Xylose - Lysine Lactose Bile Salts Sodium Thiosulfate Yelrow Transparent on Deoxycholate) Sucrose Red Medium Xylose (H:zS+ = {;Jlack) Enrichment Broths = Selenite and GN Broth  Enterobacteriaceae: Primary Differentiating Tests TSI Ornithine VP Urease Lysine Motility H2S Deaminase DNAase Citrobacter K(A)/A, Gas V - V - + V - - Enterobacter A/A, Gas + + - V + - - - Escherichia - A(K)/A, Gas V - + V - - - Klebsiella A/A, Gas - + V + - - - - Morganella KIA, Gas + - + V + - + - Proteus KIA, Gas - - V + + + + V Providencia KIA, Gas - - V - + - + - Salmonella KIA, Gas - - + + + + - - Serratia K(A)/A V + - + + - - + Shigella KIA V - - - - - - - Plesiomonas KIA + - - + + - - - v = variable I * oxidase positive Shaded areas = Key reactions differentiating similar genera I · 1tsrP_e~r ~ ~ & - ~ --L) ,., "" ~ ,_ _.,.. \0 150 Klebsiellae Citrobacter 1. Opportunist; UTI , pneumonia ; 1. Opportunist ampicillin-r esistant 2. Lysine negative 2. Kleb siella a. Non-motile 3. Similar to Salmonella biochemically b. Has capsule Proteus c. Ur ea variable 1. Key Characteristics d. Ornithine negative a. Urea positive e. VP positive f. Can cause lobar pneumonia b. Deaminase positive g. New member: K. granulomatis 2. P. mirabilis ❖ Formerly Ca.lymanatohactedum a. Most sensitive to penicillins ❖ STI b. Indole negative 3. Enterohacter 3. P. vulgaris a. Motile a. Indole positive b. Ornithine positive b. H2S positive 4. Serratia Yersinia a. May produce r ed pigment 1. Y. enterocolitica b. DNase, gelatinase positive a. Optimal growth = RT; cold enrichment c. VP positive b. Invasive and toxigenic Klebsiella 2. Y. pseudo tuber culosis - Acute mesenteric pn.eumoniae lymphadenitis and "pseudotuher cules" Kleb pneumo in 3. Y. p estis - bubonic plague lungs, where Plesiomonas else would I be? 1. Oxidase positive Maybe in a wound, bladder 2. Reclassified into E11ter ohacteriaceae or kidney. 3. Lophotrichous flagella I hove no flagella and I can't swim. 4. Associated with diarrheal disease Test this out with S-1-M. BIOCHEMICAL TESTS I'm blue on citrate, prefer lysine. 1. Oxidase test Maybe pink on urea, hate ornithine. a. Reagent tetramethyl p- Salmonella phenylenediamine dihydrochloride b. Positive = purple 1. Large number needed for infection (> 100,000) 2. Nitrate test a. Reagen ts - a-naphthylamine, 2. Biochemical r eactions sulfanilic acid a. H2S positive b. Positive= pink (use zinc powder to b. Lysine positive confirm) c. Indole negative d. Urea negative 3. ONPG test a. Detects B-d-galactosidase 3. S. enterica- >2000 serot yp es including b. Reagent - O-nitrophenyl-B-d- a. S. cholerasuis - septicemia galactopyranoside b. S. typhi - typhoid fever c. Positive = yellow ❖ B.lood positive early - 1st week ❖ S too.l positive late- 2nd-3rd week 4. TSI (triple sugar iron agar) slant c. S. arizon e - ONPG pos (other s n eg) a. 0.1% glucose, 1% sucrose, 1% lactose b. Yellow butt - glucose fermented c. Yellow slant - lactose or sucrose fermented  151 d. Red slant - neither lactose nor ❖ Also tests for motility and indole sucrose fermented (Kovac's) e. Black butt - H2S produced 8. Indole test 5. Kligler Iron Agar (KIA) - same as TSI a. lndole split from tryptophan but with only glucose, and l actose, no b. Reagent (Kovac 's) - p-dimethyl- sucrose aminobenzaldehyde c. Positive = pink 6. Citrate test a. Media green 9. Urease test b. Positive = blue a. Urea hydolyzed to ammonia and CO2 7. Decarboxylase tests b. Phenol red indicator turns pink if a. Measures ability to decarboxylate positive amino acids ❖ Lysine +- Cadaveri.ne 10. Voges-Proskauer (VP) ❖ Ornithi.ne +- Putrescine a. Detects acetylmethylcarbinol ❖ Arginine +- Putresci.ne (acetoin) ❖ Indicator dye = bromcresol purple b. Reagents= KOH and a-naphthol b. Lysine iron agar (LIA) c. Positive = pink ❖ Has H2S indicator ❖ 0.1 % glucose and 1% lysine ❖ Positive = purple butt ❖ Slant of LIA turns red for lysine deaminase c. Motility-indole-ornithine (MIO) Breakdown Products of ❖ 0.1 % glucose and 1% ornithine Amino Acids ❖ Positive = purple butt ((A)\ REMEMBER! !:~:::!:~~7':;~:e (TSI Reactions) ~ The yellow sun shining over the hot desert = A/A. The moon shining over the desert = KIA. The moon over the mountain = K/K. AJA KIA, H2S + KIA K/K (Sun over Desert) (Moon over Desert) (Moon over Desert) (Moon over Mountain) ~a~ ~ :.M-f· ·'-~· ·~ ,:.. ··. -~;:,,;:.'~.?,· :'.,.r·... ·~' ,. *}) /,/I\'...!' , I tf.P~ p seudomonas ~ ~~.,. Pseudo"moon"as FIRECRACKERS S higella Ci trobacter S erratia C itrobacter C itrobacter P rovidencia E scherichia A rizona A rizona P lesiomonas E nterobacter P roteus S almonella P roteus K lebsiella Wear a cap to E dwardsiella Seek shelter in the protect yourself A case of fire- Y ersinia from the geyser bot desert. (H2S +). crackers going off Sk(c)ipppy Coyote smells like sulphur. howls at the moon. (H2S +). 152 TS/ - Biochemical Reactions A/A K/A K/K Escherichia Proteus Salmonella Shigella Pseudomonas (indole+) (urea+, deam+) (ma/-, ONPG-) (citrate-, non-motile) (ox+, blue-green pigment, growth at 4 2 "C, growth in Enterobacter Arizona Citrobacter Providencia cetrimide) (ODC+, sugars) (LDC+) (LDC-) (deam+) Other non-fermenters Klebsiella Citrobacter Edwardsiella Citrobacter (ODC-) (LDC-) (indole+, LDC+) (citrate+) Serratia P/esiomonas (sugars) (oxidase +) Proteus (urea+, deam+) Yersinia (small colonies, urea+, dea,n-) Positive Reactions in Biochemical Tests Enterobacteriaceae - A/A, Gas © 0 Urea I I lndole 0 © Phe deaminase 0 I I I I © Citrate 0 © LDC 0 © I lndole 0 I I © LDC 0 I I Citrobacter I E.coli © ODC 0 Proteus Proteus Arizona Citrobacter vulgaris mirabilis (H,S+) I Enterobacter I Enterobacter agglomerans © ODC 0 © I Gelatin 0 I Klebsiella Biochemical Reactions of I Serratia I Lactose Ent.erobacteriaceae © Citrate 0 I Enterobacter I Hafniae aerogenes a/vei  153 Enterobacfertaceae - K/A, H2S+ EB - - - - - - Urea or Phenylalanine deam - - - - - - 0 (PDA) EB I lndole 0 @ I lndole 0 I Proteus vulgaris I Proteus mirabilis Ed C 7 @ LDC 0 CitrobactJ, freundii 0 ODC EB l Salmonella typhi I Salmonella sp (most subgroups) 11. Phenylalanine deaminase ❖ Pigm en t production a. Reagent = ferric chloride l@j' Pyocyanin (blue green ) h. Positive= green 11'.i" P yoruhin (rust) GRAM NEGATIVE NON-FERMENTERS 11'.i" P yoverdin (fluorescent) g. Drug resistant 1. Oxidase- positive (some exception s, i. e. h. Infections- burns, pneumonia, Stenotrophomonas) swimmer's ear, eye infections, UTI 2. May not grow on MacConkey's agar 5. Burkholderia cepacia 3. Glucose NOT fermented a. Oxidase weakly positive a. TSI= alk/alk b. Growth on MAC b. Oxidative Fermentative (OF ) media c. Oxidizer ( OF) ❖ Semi-solid d. Polymyxin B r esistant ❖ Low protein e. Opportunistic pathogen ❖ Bromthymol blue indicator ❖ Serious infection in CF patients ❖ Incubate tubes in pairs ❖ Yellow-brown insoluble pigment 11cW Open- oxygen 11cW Closed - oxygen-restricted i& Pair of tubes yellow = fermenter 11cW Pair of tubes green = asaccharolytic (non-utilizer) 11'.i" Open tube yellow and closed tube green = oxidizer 4. Pseudomonas aeruginosa a. Most colOJllon b. Oxidase positive c. Growth on MAC (lactose n egative) d. Oxidizer (OF) Organisms Associated witb e. Polymyxin B susceptible Cystw Fibrosis f. Opportunistic pathogen ❖ Mucoid in CF patients 154 Gram Negative Oxidase Positive Fermenters VIBRIO 5. Aeromonas 1. Characteristics a. Motile by pola r flagella a. Curved rods with polar flagella b. Most are indole positive b. TCBS selective for vibrios c. Growth on MAC c. BAP - hemolytic colonies d. Cellulitis, wound infections and d. Lactose negative ( differs from diarrhea enterics) e. N03 positive (NO3 to NO2 ) OTHER ORGANISMS 2. V. cholera (Serogroup 01) 1. Campylohacter jejuni a. Gastroenteritis - rapid onset 3-10 a. Small curved rods, "seagull hrs.; profuse diarrhea appearance" (light staining) b. Need high concentration of b. Microaerophilic, 2-4 days for organism unless hypochlorohydric growth c. Stools contain mucu s fleck s c. Growth on CAMPY agar (described as "rice water" stools) d. Found in raw poultry and d. Yellow on TCBS contaminated water (like Salmonella) 3. V. parahemolyticus e. Erythromycin or tetracycline for a. Green on TCBS treatment b. Enteritis f. Bioch emical reactions ❖ Catalase positive 4. V. vulnificus ❖ Oxidase positive a. Gr een on TCBS ❖ Hippurate positive b. Septicemia - can kill immunocompromised or diabetics 2. Helicobacter pylori a. Associated with gastric and duodenal ulcers b. Produces large amounts of urease Campylobacter jejuni Just call me Jay June Eye. I might be in water or chicken pot pie. I like just a touch of o2 More oxygen and I'll be through! Campy-plates are best to grow _ At 42 degrees or so If I grow, Gram stain me And curved rods are what you'll see.  155 Zoonotic Diseases (Acquired Directly or Indirectly from Animals) ORGANISM DESCRIPTION DISEASE NOTES Bruce/la Gram Neg Cocoobacillus Brucellosis Blood Culture Pos in First Two Weeks (Hold 21 days) ~ CO2 H 2S Thionine Basic Fuschin 8. abortus (cows) + + + - 8. suis (pigs) - +/- - + 8. melentensis (goats) - - - - @ ' REMEMBER! Brucella R....... Cows eat Thionine. Pigs eat Basic Fuschin. Goats eat neither. ~ ORGANISM DESCRIPTION DISEASE NOTES Francisefla tularensis Faintly staining Gram Neg Coccobacillus Tularemia Infected by Tick Bite Tiny Pinpoint Colonies "Rabbit Fever" High Risk to Lab Personnel.,, ~ Cystine-Glucose Media Francis H2S + with Lead Acetate Biosafety level 3 I the Rabbit \ Uses the _ 'rt· Hood._(' Yersinia pestis Gram Neg Plague Yersinthe Safety Pin J Bi-Polar Staining (resembles safety pin) Transmitted by Fleas, Rats, Other Mammal Reservoirs Biosafety level 3 Pasteurefla multocida Gram Neg Rod Contracted from "Mousy" Odor Bi-Polar Staining Cat and Dog Bites Oxidase and lndole Positive Biosafety level 2 ~ Ferments Glucose and Sucrose Streptobaci//us moniliformis Long Filamentous Gram Neg Rods with "Rat Bite Fever" Ascitic Fluid Sample Needed; Swellings (pleomorphic); "Puffball" or Haverhill Fever Sodium Polyanethol Sulfonate Ji "String of Pearls" Colonies in (SPS) Inhibits growth Thioglycolate Broth 156 Fastidious Gram Negative Rods GENERAL INFORMATION 1. Source - mouth flora Bordetella pertussis 2. Pathogenic in immunocompromised hosts; causes: Pertussis a a. P eridontal and jaw abscesses fancy name for (Eiken ella , Aggr ega tihacte1·, whooping Capnocytophaga) cough. b. Infectious endocarditis Take the c. Bacterernia ( Capnocytophaga, vaccine to be Cardiobacteri um) well off. Bordet-Gengou or 3. Grow slowly and require 5 - 10% CO2; NO growth on MAC Regan Lowe Are the best for me to grow. CAPNOCYTOPHAGA It may take many days to see 1. Capnophilic A mercury droplet colony. 2. F usiform shape HAEMOPHILUS INFLUENZAE 3. Bacter ernia/ septicemia 1. Sma ll, non-motile gram negative r od 4. Gliding motility BORDETEll.A PERTUSSIS 2. Requires growth factor s a. X factor = hemin 1. Gram nega tive coccobacilli; causes b. V factor = NAD "whooping cough" c. Both factors found in blood, but n eed h eat to break down red cells and 2. Requi r e special media release factors, so chocolate used a. Classic = Bordet-Gengou (potato infusion wi.th glycer ol and 20% d. Satellitism ❖ S. aureus produces V factor and SRBc's) and penicillin b. Regan/ Lowe (oxoid char coal agar , releases X factor by hemolyzing blood ❖ Haemophilus will grow in the 10% horse blood, cephalexin); hemolytic zone surrounding staph as lon ger shelf life satellite colonies on blood agar plate 3. Old m ethod for collection - cough plate; ❖ Need 5% h orse or rabbit blood to better to collect NP swab and plate see hem olysis directly; P CR to confirm ID e. Infections ❖ Meningitis - 4. Colony I& 2-4 year s a. Incubate 72-96 hrs, 35°C I& Preceded by n asopharyu geal b. Pinpoint, "mercury droplet" colonies colonization and hacter ernia HACEK GROUP OXIDASE CATALASE NITRATE INDOLE NOTES Aggregatibacter aphrophi/us - / weak+ - + - Colonies similar to (Haemophilus ) Aggregatbacte r ; Endo carditis A ggrega tibacter actino- +/- + + - Peridontal & Jaw A bsc esses; mycetemcomitans High Number in Plaque; Center (Actinobaci/lus) of Colony has 4 -6 Pointed St ar Cardiobacterium hominis + - - + Can Cause Endocarditis; Can Give False Positive Gram reac- tions Rosette g rouping Eikenella corrodens + - + - "Bleachy" Odor; Pits Agar; Three Zones of Growth; Peridontal and Jaw Abscesses Kingella kingae + - - - Septic A rthiritis ; Osteomyelitis in Children ; Spreading Corrod ing Colonies  157 ❖ Epiglottitis MYCOPLASMA AND UREAPLASMA I@' Ages up to 2 years; do NOT 1. Smallest free-living microorganisms collect throat culture (blood (125-250 nm) culture best) I@' May require intubation 2. Lack cell wall (bound by single triple ❖ Pneumonia 60 years layered membrane) ❖ Conjunctivitis (''pink eye") I@' Very contagious 3. Does not stain with Gram's stain; can ❖ Cbancroid - H. ducreyi use Dienes stain I@' Painful genital ulcers or soft 4. Center of colony grows into special chancres media ( contai.11s sterols) giving ll'.ii1f Gram stain of drainage appearance of inverted "'fried egg" shows tiny gram negative coccobacilli with a "school of 5. M. pneumoniae fish" arrangement a. Primary atypical pneumonia or "walking" pneumonia 3. Characteristics b. Causes positive cold agglutinin titer a. Growth only on chocolate, NOT blood agar plate ( unl.ess mixed with (> 1:32); false positive RPR c. Treat with erythromycin or S. aureus) tetracycline b. Use X and V discs on Mueller- Hinton agar to detect growth 6. Ureaplasma urealyticum requirements a. Non-gonococcal, non-chlamydia! c. Susceptibility zone interpretations urethritis, especially in males differ from conventional K- B zones b. Produces urease ( use Haemophilus test media c. Treat with tetracycline or (HTM), supplemented Mueller- spectinomycin Hinton agar and incubate in 5-10% CO2) 7. M. hominis d. Perform beta lactamase to a. May colonize GU tract; post partum determine sensitivity to ampicillin; fever cefuroxime, ceftriaxone, cefotaxime b. Tetracycline; resistant to (meningitis) erythromycin (all other Mycoplasma l£GIONELLA PNEUMOPHILA are sensitive) 1. Legionnaires' disease - severe SPIRILLACEAE pneumonia; Pontiac fever = milder 1. Rigid, helically curved rods with one or form more turns; corkscrew motility by polar flagella; gram negative 2. 75% illness due to L. pneumophila serogroups 1 and 6 2. Spirillum minor - "rat bite" fever a. Visualize by darkfield or stain with 3. Sources - potable water, faulty air Giemsa conditioner vents, lakes and ponds b. 2-3 spirals and bipolar polytrichous tufts of flagella 4. Identification c. No growth on artificial media a. Specimen - sputum, bronchial washing, pleural fluid, lung aspirate or biopsy b. Growth on BCYE (buffered charcoal yeast extract) with or without antibiotics but not on chocolate or blood X ud V Factors 111Jd c. Direct exam - Giemsa and Gram Susceptibility Testing for stain with basic fuchsin counter Haemophilus stain Isolation of Legioaella 158 O'fher Fastidious Gram Negative Rods ORGANISM DESCRIPTION DISEASE NOTES Bordetella pertussis Gram Neg Coccobacillus; Pinpoint, Pertussis ( Whooping Bordet-Gengou Media; NP Swab "Mercury Droplet" Colonies Cough) and Plate Directly Haemophilus Small, Non-Motile Gram Neg Rods H. influenzae - Causes Require X and V Factors H. ducreyi - "School of Fish" Influenza, Meningitis, and Epiglottitis H. ducreyi - Causes Genital Ulcers Legionella pneumophilia Growth on BCYE Legionnaires' Disease No Growth Routine Media Mycoplasma/Ureaplasma Colony Appears as Inverted Fried Egg M. pneumoniae - Dienes Stain NOT Gram Stain Causes Primary Atypical Pneumonia (-i-Cold Agglutinin Titer)