Review Notes in Microbiology - MTAP - NU-Manila PDF

REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA BASIC INFORMATIONS GENERAL Prokaryote, CHARACTERISTICS With both RNA & DNA OF BACTERIA Usually with CELL WALL except: _____________________...

REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA BASIC INFORMATIONS GENERAL Prokaryote, CHARACTERISTICS With both RNA & DNA OF BACTERIA Usually with CELL WALL except: _________________________________________ mycoplasma & ureaplasma (no gram staining) Some can produce spores _______________________________________________ bacillus & clostridium (anaerobes) TOXINS biologically Exotoxins Produced by gram (+) except ________________________, Listeria may also be produced by gram (-) produced PROTEIN in nature; unstable to heating excreted by living bacteria poisons LIMULUS LYSATE TEST - detect Examples: _____________________________________________________________________________________________ TSST - S. aureus; Diptheria Toxin - C. diphtheria; botulinum toxin - C. botulinum coagulase - enzymes endotoxins in body fluids Endotoxins virulence factor S. aureus Usually produced by gram (-) organisms __________________________________________________________________ shigella spp. uses: Aq. extract of horse shoe crabs Example: _____________________________________________________________________________________________ shigella spp. released only after cell death (+) clumping LPS in nature BACTERIAL CELL CELL WALL Main component is _______________________________________ peptidoglycan Shapes: round - cocci, rod - bacilli; spiral STRUCTURE a.k.a. murein layer/ Give shape to the organism; Provides attachment for flagellum cell wall inhibitor - kill bacteria using cell wall peptidoglycan layer Usual site of antibiotic action, basis of gram staining Also known as _____________________________________________________ peptidoglycan layer Disaccharides: N-acetyl-glucosamine & N-acetyl-muramic acid gram (+) With Teichoic acid, thicker peptidoglycan layer gram (-) No teichoic acid, thinner peptidoglycan layer, with outer membrane, LPS CAPSULE Function: _______________________________________________ anti-phagocytic = virulence factor G(-) organism Responsible for _________________________________colonies mucoid Capsular swelling test _________________________________________ NEUFELD QUELLUNG SLIMY AREA COVERING THE With POLYSACCHARIDE CAPSULE: S. pneumoniae, K. pneumoniae, N. meningitidis CELL WALL Glycocalyx – less organized compared with capsule, slimy layer, polysaccharide containing i.e., S. mutans PILI Usually found in gram negative organisms Fimbriae adherence/ attachment (virulence factor) e.g. N. gonorrhoeae a.k.a. G(-) org. COMMON PILI __________________________________________________ SEX PILI – for gene conjugation, transfer of genetic material e.g. E. coli ENDOSPORES Target of sterilization, contains DIPICOLINIC ACID/calcium dipicolinate autoclave - method of destruction for bacteria or SPORES Enables organism to withstand injurious conditions resistant structure w/ spores C. tetani Bacillus & clostridium WITH TERMINAL SWOLLEN SPORES ____________________________________ In B. anthracis spores are ________________ central while in C. botulinum __________________ subterminal w/ polypeptide D-glutamic acid: B. anthracis SCHAEFFER & FULTON METHOD: used primary dye (malachite green) and counter stain (safranin) w/ hyaluronic acid capsule: P. multocida result: spores = green; others= red polyribosul ribitol phosphate: H. influenzae alginate capsule: P. aeruginosa pg. 1 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA FLAGELLA Organ for locomotion Most cocci are Non-motile, Bacilli and Spiral organisms are usually motile # of flagella: In Spiral organisms it’s Methods to detect Motility 1) atrichous - no flagella 2) monotrichous - single flagellum at 1 end called 1) HANGING DROP presumptive/ initial test for Listeria 3) amphitrichous - single flagellum at both AXIAL FILAMENTS or 2) Use of Flagellar stains: Grays, Leifson ends _______________ periplasmic flagella 3) Use of SEMI-SOLID MEDIA ___________________________________ SIM 4) lophotrichous - tuft/ bundle of flagella at _______________ non motile (-) 1/both ends ________________________if growth is AT THE LINE OF INOCULATION 5) peritrichous - surrounded w/ flagella types of motility ________________________if motile (+) growth is OUTSIDE THE LINE of INOCULATION 1. True motility - Motility is best observed at __________________________ room temp presence of flagella Tumbling motility L. monocytogenes Twitching motility K. kingae 2. Brownian movement -movement of Gliding/sliding C. gingivalis non-motile cause by the motility movement of molecules Darting motility Campylobacter spp surrounding the org. Shooting star motility V. cholera Corkscrew motility Spiral organism/ spirochetes BACTERIAL CELL INCLUSION BODIES Metachromatic granules-BABES ERNST BODIES-Volutin seen in STRUCTURES stored food _________________________________________________________ C. diphtheriae MUCH granules __________________________________________ M. tuberculosis Halberstaedter Prowazek -glycogen containing seen in ______________________ C. trachomatis Other structures CELL MEMBRANE – phospholipid bilayer, No STEROLS except in _______________________________ Mycoplasma NUCLEOID – contains the genome RIBOSOME – mediates protein synthesis SPECIMENS, Important Rules When to collect specimen _____________________________________________________________________________ during acute phase of infection/ febrile stage (w/in 72 hrs) SPECIMEN Observe _________________________, aseptic technique collection containers must be properly labeled & must be sterile COLLECTION & Adequate amount must be collected. HANDLING Collected samples must be transported without delay ______________________________________________ 30 mins - 2hrs In the processing of samples, observe LEVEL of PRIORITIZATION biochemical testing PRIORITIZATION of SPECIMENS susceptibility testing 1 Critical / Invasive lumbar tap CSF, Amniotic fluid, blood, pericardial fluid, heart valves 2 UNPRESERVED feces, sputum, wound drainage 3 Quantitation required Catheter tip, urine, tissues for quantitation 4 Preserved urine, feces, swab in holding media 5 Batch Processing Sputum, AFB culture BLOOD Purpose of blood culture _____________________________________________________________________________ rule out septicemia (many bacte in blood) / bacteremia (mere presence of bacte in blood) Blood pathogens ____________________________________________________________________________________ E. coli, S. aureus (common cause of sepsis) pg. 2 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA Biomarker for fungemia ____________________________; C. albicans potential biomarker for sepsis ______________________ Procalcitonin Phlebotomy site must be cleansed with ________________________________________________________________ 70-95% OH - iodine scrub (60 secs) - OH rinse Adult - 20 mL both sites with 1 hr. interval Common contaminants ______________________________________________________________________________ S. epidermidis, Viridans spp. Dilution of blood to medium __________________________________________________________________________ Anticoagulant ______________________________________________________________________________________ 0.025% SPS disadvatage: inhibitory action to Neisseria & G. vaginalis; remedy: add 1% gelatin ___________________________________________________________________________________________________ 2. Endotracheal Not to be used for bacteriologic culture ________________________________________________________________ EDTA & citrate molecular test (PCR) - white top tube w/ EDTA aspirate - spx to be Anticoagulant for viral culture but inhibitory to gram + organisms & yeast ___________________________________ heparin collected from a px on a ventilator/ intubated Routine blood culture bottles are held for 7 days For Brucellosis detection ________________________for 3-4 weeks leptospirosis detection ______________________________ 8 weeks neonatal - S. agalactiae CSF detect meningitis Collect in 3 tubes, use tube ______________ 2 for micro adult (>29y/o) - S. pneumoniae 1st priority (5-29) N. meningitidis Storage temp ______________________ 37C transport temp _____________________________ room temp 100,000 = UTI colony counting Formula: # of colonies counted x dilution factor = colony count/ml of urine Dilution factor if 1 ul loop was used ________________________if 1000 10 ul loop was used ________________________ 100 SPUTUM non-sterile Evaluate quality, Do Bartlett’s Classification ____________________________________ sputum or saliva TB = M. tuberculosis detect Respiratory tract infection If more than 10 SECs and less than 25 PMNs were noted ___________________________ saliva (SEC) sputum = alveolar macrophage slow growers For M. tuberculosis identification, we do ________________________________________________________________ digestion + decontamination Purpose of decontamination __________________________________________________________________________ remove contaminants & normal flora BSL 1= Minimal threat Purpose of Digestion ________________________________________________________________________________ liquefy the sample - freeing the trapped org. BSL 2= moderate/ ingestion (EX. SAL & SHI) - Gold standard for digestion & decontamination _________________________________________________________ NALC (DIGEST) + NaOH (DECONTAMINANT) GIT pathogens Other agents: N-ACETYL- L CYSTEINE) BSL 3= highrisk/ inhalation BSL 4= Extreme risk/ life ✓ Z-TSP (zephiran & Trisodium phosphate) threatening diseases (EX. EBOLA) ✓ 4% NaOH commonly used (decontaminant & digest) ✓ Cetylpyridium chloride Sodium chloride method ✓ 5% oxalic acid ______________________________________________________________________________ for specimen likely to contain P. aeruginosa M. tuberculosis is classified as a BSL ______ 3 pathogen and therefore culture must be done using BSC Class ______ 2 THROAT SWAB Identification of ________________________________which S. pyogenes is considered as the major throat pathogen detect Diphtheria Normal throat flora _________________________________________________________________________ Viridans strep pg. 3 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA NASOPHARGYNGEAL Nasopharyngeal swab may be required to detect carrier state of ______________________________ N. meningitis (w/ or w/o capsule) and also methods to identify Bacte spp. SWAB identification of B. pertussis & H. influenzae B. Pertussis causes whooping cough 1. Genotypic- molec tech For bacterial culture use Dacron, Calcium alginate. Toxic to Neisseria ____________________________________ cotton swab (fatty acids) 2. Phenotypic - observable char. microscopic app., For Viral culture, Dacron, Cotton, Rayon fibers. Toxic to viruses _________________________________________ Ca alginate (prevent viral replication) biochem, colony app, STOOL Detection of GIT pathogens like ________________________________________________ salmonella & shigella Enterobacteriaceae fam - EMB, MAC, SSA Maybe collected if stool collection is not possible _________________________________ rectal swab # of quadrants streaked on plated media ________________________________________ 4 (semi quantitation) Gram staining is not usually done STAINING OF direct staining Use of only 1 dye, the color of the dye is the resulting color i.e. methylene blue BACTERIAL CELLS indirect/ negative/ relief staining Only the background and not the organism is stained i.e. india ink Organism appears colorless SPECIAL STAINING Use to demonstrate special features of the cell Capsular stains Hiss, Anthony’s, Tyler, Muir Spore stain Dorner’s, Schaeffer and Fulton, Wirtz and Conklin Flagella Grays, Fisher and Conn, Leifson Metachromatic granules Albert’s, Neisser, Ljubinsky, Ponder,methylene Blue, Lindergran, Burke’s technique Polar Bodies Wayson stain, Methylene Blue spirochetes Levaditi’s DIFFERENTIAL To differentiate one organism from another STAINING Examples: _____________________________________________________________ gram stain & Acid Fast stain GRAM STAINING PURPOSE Reagent Gram Positive Gram Negative VIAS Primary Stain/ Initial violet stain crystal violet violet Mordant violet grams' iodine (alk) violet Decolorizer OH, acetone / OH + acetone mixture colorless violet Counterstain Secondary stain safranin violet red GRAM POSITIVE: _________________________________GRAM violet NEGATIVE: __________________________________ red Most Critical step in gram staining ________________________________________________________ decolorization Hucker’s modification __________________________________________________________________ gram staining procedure for fungi G(+) (ammonium oxalate + crystal violet) Carbol fuchsin can be used as counterstain in place of safranin. This may be done to improve staining of some gram-negative organisms that are poorly stained with safranin Rules: pg. 4 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023 NOTE: Genus Chlamydia / Chlamydophila Elementary Body - “Infectious form” Reticulated Body - “Reproductive form” REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA cell culture: McCoy Cells & Mela Cells MORAXELLA 1. All COCCI are gram (+) except Neisseria, Branhamella and Veilonella 2. All BACILLI are gram (-) except: Mycobacterium, Corynebacterium, Clostridium, Bacillus, Erysipelothrix, Lactobacillus, Listeria 3. Higher forms of organisms like Actinomyces, Nocardia, Streptomyces, yeast and molds are gram (+) 4. All Spiral organisms are reported as Gram (-) Not GRAM stained are Rickettsia; Chlamydia (intracellular); Mycoplasma; Ureaplasma (wall less); spirochetes (can’t resolved by bright field) ACID FAST STAINING Purpose Ziehl Neelsen / Hot Kinyoun’s / Cold method Auramine-Rhodamine most for bacteria with mycolic acid method DSSM (direct sputum smear microscopy) AFB in tissues (Fluorochrome) sensitive SIZE: 2X3 cm Primary carbol fushin carbol fushin auramine rhodamine Mordant heat wetting agent ( tergitol) X acid OH = HCL + ETOH Decolorizer 3% acid OH 3% acid OH 0.5 % acid OH ZIEHL/KINYOUN - REPORTING phenol = accentuator # of AFB Report Counterstain 0 No AFB seen in 300 visual fields Methylene blue Methylene blue 0.5% KMnO4 +n 1-9/100 fields Result Acid Fast: _________________________________ red against blue 1+ 10-99/100 fields AFB = yellow against black bg 2+ 1-10/OIF (at least 50 fields) Non-Acid fast: ______________________________ blue/ green 3+ >10AFB/OIF (at least 20 fields May be used as substitute for Adv: will allow examination under LPO malachite green FLOUROCHROME - REPORTING Methylene Blue Counterstain easier to read bcoz of better contrast 0 negative 1-2 AFB/70 fields doubtful (repeat w/ new spx.) 1-2 AFB/100 OIF 1+ ACID FAST ORGANISMS – are organisms that are very hard to stain but once stained they are difficult to decolorize due to 2-18 AFB/ 50 fields 2+ 4-36 AFB/ field 3+ MYCOLIC ACID / HYDROXYMETHOXY ACID that envelopes the bacteria >36 AFB/field 4+ Rule: All bacteria are Non-Acid Fast except: MYCOBACTERIUM, slightly acid fast is NOCARDIA CULTURE & culture media Any medium that contains all what is needed to support bacterial growth CULTURE MEDIA culture Growth of organism in culture i.e Pure culture, mixed culture, stock culture Types as to LIQUID SEMI-SOLID SOLID BIPHASIC consistency Solidifying agent/ agar 0% 0.5-1% 2-3% both solid & liquid Examples TSB SIM Liquefiable: HBT broth water infusion Alkaline EMB human blood Bilayer Peptone MSA (G. vaginalis) Water Castanedals (brucella) BHI Non-liquefiable: Rice Medium for fungi Types as to purpose General Purpose Contains only what is needed to support bacterial growth routine cultivation )e.g. nutrient broth, nutrient agar Enrichment media To enhance bacterial growth increase bacterial yield e.g. alkaline peptine water = vibrio selenite broth, tetrathionate broth = G(-) Enriched media For growing fastidious organisms like unable to grow in ordinary media e.g. Neisseria, Haemophilus Contains blood e.g. BAP and CAP pg. 5 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA Selective media Used to promote growth of a particular organism & at the same time preventing growth of other cystine tellurite blood agar (C. diphtheriae) inhibitor = potassium tellurite thayer martin (Neisseria) inhibitor: vancomycin (G+), colistin (G-), nystatin (fungi) It contains inhibitor Differential Media Used to differentiate organisms that are growing together MAC & EMB - differentiate Lactose fermenter from non NLF - colorless; LF- pink-purple Selective & Differential Examples: MAC & EMB = (G-) bacilli Transport Media Purpose: maintain viability of organism during specimen transport JEMBEC & TRANSGROW - Neisseria ; Cary Blair - stool pathogen (vibrio) ; Amies - respiratory spx JEMBEC, Cary Blair, Transgrow, Amies Biochemical test media Used for biochemical testing which aids in bacterial identification identify spp. Media for susceptibility test MHA Motility Test Medium SIM BACTERIAL MANUAL OBSOLETE IDENTIFICATION SEMI-AUTOMATED i.e API- Analytical Profile Index – uses plastic strips & microtubes with biochemical substrates METHODS the biochemical substrates are inoculated with pure culture suspension API 20E _________________________________API enterobacteriacea 20A __________________________________________________ anaerobic AUTOMATED VITEK, MALDI-TOF more rapid automation program for bacterial ID and susceptibility STEPS IN PCR 1. Denaturation __________________________ 2. Annealing ___________________________3. Extension _______________________ SIGNIFICANT GRAM (+) COCCI & GRAM (-) COCCI IDENTIFICATION CATALASE TEST To differentiate Staphylococci & Micrococci from Streptococci breakdown of water and oxygen TESTS FOR Reagent __________________________________ 3% H2O2 (+) result ________________________________________________ vigorous bubbling STAPHYLOCOCCI & Use of colonies from BAP _____________________________________________________ false (+) MICROCOCCI MODIFIED OXIDASE For identification of ___________________________________________ micrococci SBA, MSA, CNA, PEA TEST / MICRODASE Uses tetramethyl-p-phenylenediamine dihydrochloride in DMSO (dimethylsulfoxide) 6% oxidase rgt (+) result ______________________________________________ blue color COAGULASE TEST To differentiate the pathogenic S. aureus from other Staphylococci definitive test for S. aureus Methods: Slide ______________________________________ detect bound coagulase (clumping factor) Tube __________________________________________ detect free coagulase Uses Rabbit’s plasma collected using EDTA ____________________________________________________________ Use of Citrated Plasma _______________________________________________________________________________ false (+) (+) result: __________________________________________________________________________________________ clot formation Tube method will require incubation at 37 deg for 4 hrs, if (-) incubate further at RT for 16 hrs MANNITOL Maybe carried out to detect S. aureus which is (+) FERMENTATION Required media ____________________________________ MSA TESTS Inhibitor _____________________________pH 7.5% NaCl indicator ______________________________ phenol red (+) result: development of yellow halo around the colonies (-) red pg. 6 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA A (+) result would mean that (1) the organism was able to ferment mannitol and (2) the organism was able to tolerate high salt concentration DNase test May be carried out to detect the following organisms ________________________________________________________ (+) SMS: S. aureus, Moraxella, Serratia 2 methods: HCl precipitation method – uses ___________________________(+) DNase agar result clearing of agar around the colonies _______________________________________ rgt: 0.1 Normal HCl Dye method – involves incorporation of dyes to medium, methyl green or toluidine blue (+) result with methyl green _________________________ clear zone around the colonies with toluidine blue _______________________ pink zone around the colonies Cephalosporinase test Purpose _______________________________________________________________________________________________ detect beta lactamase production Or Beta Lactamase test Uses _________________________disk cefinase with substrate ________________________________________________________ nitrocefin (+) result ______________________________________________________________________________________________ pink to red (+) catalase; (-) coagulase, NOVOBIOCIN DISK Differential test for S. saprophyticus and S. epidermidis mannitol, Dnase TEST S. saprophyticus __________________ resistant S.epidermidis _______________________ sensitive S. aureus - sensitive Staphylococcus - Facultative anaerobe Micrococcus - Strict Aerobe Aerobic growth positive positive Anaerobic growth positive negative Lyostaphin Susceptibility sensitive resistant Modified Oxidase Test negative positive MICRODASE DISK negative positive Bacitracin Susceptibility resistant sensitive Furazolidone /Furoxone Susceptibility sensitive resistant Catalase Test positive positive Benzidine Test negative Positive Glucose Utilization/ OF medium fermentative oxidizer Hemolysis on BAP S. aureus – Beta hemolytic Gamma hemolytic microscope clusters tetrad pg. 7 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA CoNS - COAGULASE NEGATIVE STAPHYLOCOCCI Staphylococcus epidermidis and Staphylococcus saprophyticus S. aureus Staphylococcus epidermidis Staphylococcus saprophyticus PYR (-); Glucosidase (+) Colony yellow white white Catalase Test positive positive positive Coagulase Test positive negative negative Mannitol Fermentation positive negative negative Hemolysis on BAP beta gamma gamma NOVOBIOCIN SUSCEPTIBILITY sensitive S – more than 16 mm R – less 16 mm (5 ug) DNAse Test positive negative negative IDENTIFICATION LAP TEST Test for catalase (-) gram (+) cocci TESTS FOR Leucine Amino Substrate: Leucine-beta-naphthylamide STREPTOCOCCI Peptidase Reagent: cinnamaldehyde (+) Streptococci, Enterococci, Lactococcus, Pediococcus (+) result: ___________ red (-) no color or slight yellow (-) Aerococcus, Leuconostoc For Group A Beta Bacitracin Disk Test _________________________________________________________ sensitive Hemolytic – PYR Test – detection of enzyme pyroglutamylaminopeptidase or pyrrolidonylamylamidase S. pyogenes Uses _____________________________________________________________________________________ p-dimethylaminocinnamaldehyde (+) PYR (+) result __________________________________________________________________________________ red For Group B Beta CAMP TEST – Christie Atkins Munch Peterson Hemolytic Uses BAP as media, Known organism ________________________________unknown organism S. agalactiae ____________________ (+) result: enhanced arrow head zone of beta hemolysis Hippurate Hydrolysis Test – detection of the enzyme Hippurate hydrolase or Hippuricase In this test, sodium Hippurate is hydrolyzed into benzoic acid and glycine uses sodium hippurate broth To detect benzoic acid _________________________is ferric chloride used while to detect glycine use __________________________ ninhydrin rgt (+) result ____________________________________________________________________________________________ purple Bacitracin ____________________________ resistant For Alpha hemolytic Neufeld Quellung (+) ______________________________________________________ Capsular swelling test = (+) S. pneumoniae; (-) viridans Streptococci Bile Solubility test – required media ______________________ BAP/ broth S. pneumoniae = (+) neufeld, bile, Reagent ______________________________________________ 10% sodium desoxycholate (BAP) ; 2% sodium desoxycholate (+) result ______________________________________ lysis of colonies/ clearing (BAP) ; clearing (broth) _____________________ (S) optochin Viridans "green" = (-) neufeld, bile, (-) result ______________________________________________________________________________________________ intact colonies (BAP) ; partial clearing/ turbidity (broth) _____________________ (R) optochin Optochin Disk Test – uses TAXO P or ethylhydrocupreine hydrochloride S, pneumoniae _______________________________________ sensitive / susceptible Viridans Streptococci _____________________________ resistant Catalase (-) > LAP (+) > BAP > alpha = S. pneumoniae / Viridans group alpha = partial/ inc optochin = TAXO P determine greenish in BAP hemolysis beta = complete bacitracin = TAXO A gamma = no hemolysis pg. 8 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023 G(+) cocci a. Staphylococcus b. Micrococcus > catalase (+) c. Streptococcus > catalase (-) REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA For Gamma Hemolytic Screening Test ____________________________________ bile esculin test Media _____________________________________________ bile esculin media (slanted tube media) Streptococci Indicator ________________________________________ (+) result ____________________________________________ ferric ammonium citrate blackening of media Catalase (+). LAP (+). BAP (gamma) Differential Tests: PYR, Penicillin Disk Test, Salt Tolerance test -6.5% Group D (+) result in Salt Tolerance test ______________________________________________________ turbid ; (-) clear Non-Enterococci prepare media (nutrient broth) > add salt (6.5%) > inoculate & incubate PYR TEST SAL TOLERANCE - 6.5% NaCl PENICILLIN Enterococci Group D (-) (-) sensitive Non-Enterococci Enterococci (+) (+) resistant yellow & oily looking colonies Staphylococcus Characteristics Gm (+) cocci in GRAPE LIKE CLUSTERS, medium sized butyrous colonies aureus Colonies with odor similar to an OLD SOCK on MSA normal flora of anterior nares Catalase (+), Coagulase-Mannitol Fermentation- DNase test (+), VP (+), PYR (-) (+) in blood = sepsis Beta hemolytic on BAP Diseases Toxin Mediated: Food Poisoning, Toxic shock syndrome, Scalded skin syndrome or Ritter Disease media: MSA, BAP, CNA, PEA Non-Toxin Mediated: Boils, carbuncles, furuncles,cellulitis, wound infection, sty, impetigo identification test: coagulase, Virulence Factors mannitol fermentation, DNase, hemolysis (beta) resistant to beta lactam antiboitics destruction of RBCs prevent phagocytosis food poisoning not a virulence factor S. epidermidis Normal skin flora; Causes UTI, stitch abscess; causes Prosthetic Heart Valve infection pg. 9 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA CoNS = (-) coagulase, (-)mannitol fermentation, (-) DNase, hemolysis (gamma) CoNS – COAGULASE Virulence Factor: _____________________________________________________________ slime production/ biofilm formation > enhance attachment to plastic catheter NEGATIVE S. saprophyticus Most common cause of __________________________________________________ UTI (young female) community acquired UTI: E. coli STAPHYLOCOCCI Causes pyelonephritis and cystitis in those with indwelling catheters Differential test: white colonies uncertain virulence factor _____________________ novobiocin disk test: (S) epidermidis (R) saprophyticus _____________________ MRSA / ORSA Strain of S. aureus resistant to Methicillin, Nafcillin and Oxacillin. Resistance of Staphylococcus to penicillinase resistant penicillin is due to PBP2a (Penicillin binding protein 2 (in cell wall) encoded by _______________ mec A gene Treatment for MRSA ________________________________________________________________________ vancomycin (glycopeptide) Detection Methods (1) Use of CHROM AGAR rose - mauve colony MRSA _______________________________________________________________________________________ S. aureus - (R) penicillin, cephalosporins treatment : Penicillinase resistant drugs: methicillin, oxacillin no growth, colorless colonies, blue Non-MRSA ___________________________________________________________________________________ (2) CEFOXITIN DISK DIFFUSION TEST - Induces expression of PBP2A (3) GOLD STANDARD for MRSA detection ______________________________________ Molecular tech - PCR Smith & Brown ALPHA partial/ inc., greening alpha prime "wide zone" - when a colony is surrounded by inner a, outer B, due to prolonged refrigeration e.g. Viridans strep. Classification BETA complete C. perfringens - double/ target hemolysis (colony is surrounded by inner B and outer A) clear zone HEMOLYSIS ON BAP GAMMA no hemolysis LANCEFIELD CLASSIFICATION – based on cell wall polysaccharide antigen (common C carbohydrate) beta hemolysis Group A Characteristics Streptococcus Gram + cocci PYR (+), Bacitracin-Suceptible pyogenes Causes: Bacterial pharyngitis / Strep throat throat swab flesh eating bacterium major throat pathogen Pyodermal Infection – ERYSIPELAS skin infection scarlet fever – strawberry tongue ___________________________________________________________________________________ pharyngitis w/ rashes catalase (-) Necrotizing Fascitis ______________________________________________________________________________________________ rapid progressing skin infection LAP (+) POST STREPTOCOCCAL SEQUELAE _____________________________________________________________________________________ acute glomerulus nephritis, rheumatic fever (repeated pharyngitis) VIRULENCE FACTORS M protein found in cell wall major virulence factor; anti-phagocytic Protein F Promotes attachment to epithelial cells Streptokinase Dissolution of clot promotes fibrinolysis Hyaluronidase Spreading factor Erythrogenic or Pyrogenic Toxin causes rashes in scarlet fever STREPTOLYSIN responsible for B hemolysis Streptolysin O Oxygen labile, Antigenic developed anti-streptolysin O "ASO" Can cause sub-surface hemolysis on BAP Can cause hemolysis only when incubated anaerobically protein A - Aureus - Staphlyokinase ASO test - detects streptococcal infection (S. pyogenes) protein F - Pyogenes - streptokinase pg. 10 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023 alpha hemolytic - "greening" a. S. pneumoniae - normal flora > adult bacterial meningitis (>29 y/o), lobar pneumonia, otitis media (capsule - virulence) b. Viridans strep - major throat flora > Subacute bacterial endocarditis (SBE), dental carries - produce glucans and dextrans (promotes attachment on tooth surfaces REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA Streptolysin S Oxygen stable, non-antigenic Can cause surface hemolysis on BAP Can cause hemolysis when incubated aerobically Group B Streptococci Normal flora of female genital tract or lower GIT; causes Septicemia TODD-HEWITT BROTH - used to detect genital carriage of GBS during pregnancy LIM BROTH is supplemental media for S. agalactiae Streptococcus # 1 cause of _____________________________ neonatal meningitis virulence factor: capsule agalactiae In adults it can cause Postpartum endometriosis Group C, F & G Normal flora: Skin, Nasopharynx Gastrointestinal tract, Genital tract Group D Gamma hemolytic S. equinus, S. gallolyticus, S. infantarius, S. alactolyticus Non-Enterococci Encountered in blood cultures of patients with bacteremia, septicemai & endocarditis a.k.a. S. bovis group presence of S. gallolyticus subspecies gallolyticus, in blood cultures has HIGH CORRELATION with GIT Carcinoma/colon cancer S. equinus Enterococci most E. faecalis; E. faecium, E. durans, E. avium frequent isolate Causes UTI & wound infections hospital acquired UTI Virulence Factors: ability to grow in extreme conditions, Adhesins, gelatinase, Cytolysin GENUS NEISSERIA Characteristics Gram (-) non-motile, capnophilic increase CO2 CANDLE JAR - to supply 5-10% CO2 = colored & scented are inhibitory sensitive in cotton swab & cold temp. May require transport media fastidious org. Media prevent G(+) enriched chocolate agar + antibiotics prevent G(-) prevent fungi sulfamethoxazole prevent swarming org like Proteus antifungal allows growth of M. hominis & U. urealyticum antifungal prevent G(+) amphotericin B Screening test CYTOCHROME OXIDASE TEST Reagent: tetramethyl-p-phenylenediamine dihydrochloride (+) Result: ___________________________________________ purple Test to detect species CHO utilization test /fermentation test (glucose, maltose, sucrose, lactose) media: _____________________________________indicator_________________________________________________ CTA - cysteine trypicase agar phenol red In an acid pH (+) media will turn _________________________________________________________________________ yellow ; (-) red alk. ph aerobes requires 21% O2 & 0.02% CO2 OXIDASE TEST capnophilic requires 5% O2 & 5-10% CO2 MODIFIED - detect micrococci G(+) (used 6% oxidase rgt.) - result (+) blue CYTOCHROME - detect G(-) (used 1% oxidase rgt) - result (+) purple pg. 11 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA RESULTS in CHO UTILIZATION ACID PRODUCED FROM: TEST Specie Glucose Maltose Lactose Sucrose Fructose N. gonorrhea G + - - - - N. meningitidis GM + + - - - ONPG - G(-) enterobacteriaceae N. lactamica GML + + + - - -detect late lactose fermenter (+) ONPG _____ (+) N. cinerea - - - - - N. flavescens N. elongata N. sicca + + - + + N. mucosa N. subflava + + - V V asaccharolytic MORAXELLA - - - - - Neisseria gonorrhoeae Primary Virulence Factor: _________________________ pili -Kidney, coffee bean shaped diplococci clusters - tetrad - micrococcus intracellular w/in PMN FERMENTS ___________________________________ glucose lancet/ bullet diplo - S. pneumoniae -(-) on SBA SUPEROXOL TEST (+) ________________________________________________________________________ rapid test for N. gonorrhea, used 30%H2O2, result: (+) bubbling - collect specimen using swab made of dacron, rayon/ calcium alginate Causes: a) Gonorrhea __________________________________ STD - female (cervical swab), male (urethral swab) b) Opthalmia neonatorum – a gonococcal eye infection acquired by newborns when discharge from infected mother accumulates in their conjunctiva crede's prophylaxis/ erythromycin Neisseria meningitidis Primary Virulence factor: ___________________ capsule N. sicca - (+) sucrose, maltose, glucose - breadcrumbs like colonies -bean shaped diplococci Neufeld Quellung Test _____________________ positive MORAXELLA CATARRHALIS - (S) to SPS -(+) on SBA FERMENTS ______________________________ maltose & glucose Can be a normal flora, natural habitat is oro and nasopharynx. (Nonencapsulated strains) - morphologically and biochemically resembles Neisseria, Oxidase test (+) - as it produce 1. ENCAPSULATED Specimen to detect carrier state ______________ nasopharyngeal swab "indophenol oxidase" - pathogenic -Catalase (-), on BAP gamma hemolytic - cause meningitis (CSF) Causes: - normal flora of oro and nasopharynx but a) Bacterial Meningitis __________________ 5-29 y/o may cause otitis media (3rd most common cause 2. NON-ENCAPSULATED - does not degrade sugar (-) ASACCHAROLYTIC- normal flora b) Meningococcemia organism in blood -BUTYRATE DISK TEST/ tributyrin test (+) = rapid c) Waterhouse Friderichsen Syndrome – severe form of meningococcemia characterized by bleeding of adrenal test for Moraxella uses substrate: bromo-chloro-indolyl-butyrate; glands/DIC (+) result blue color c) PID which may cause sterility, perihepatitis or Fitz Hugh Curtis Syndrome - "hockey puck colonies" - remains intact when moved - compared with neisseria, it is tributyrin test & DNase test positive (-) growth on media for Neisseria because of colistin To collect specimen use ________________________________ Calcium alginate and cotton swab may be inhibitory to - (+) CAP/ SBA - older colonies - "wagon wheel appearance" N. gonorrhoea GRAM + BACILLI - SPORE FORMERS GENUS BACILLUS Bacillus anthracis _____________________________ non motile and __________________________________________________________ gamma on BAP - differential characteristics from other bacillus aerobic -category A bioterrorism agent forms the so called “disjointed bamboo fishing rod appearance”, Typically has _____________________________ square ends catalase (+) using 15% H2O2 -ASCOLI TEST - diagnostic test colonies with __________________________________consistency tenacious/ beaten egg white category A - easily transmitted; high mortality rate pg. 12 prepared by: DR. MA. CRISTINA LIWANAG – SEPTEMBER 2023  1. B. stearrothermophilus - biological indicator of autoclave REVIEW NOTES in MICROBIOLOGY- MTAP – NU-MANILA 2. B. subtilis var. niger - indicator of oven MICROSCOPIC APPEARANCE colonies may show swirling projections giving it a _________________________________________________ medusa head/ lion head appearance/ comet tail 1. disjointed bamboo 2. demo capsule using M' Fadyean stain forms the so-called STRING OF PEARL PATTERN ________________________________________________ (S) penicillin 3. demo spores using Schaeffer & fulton Forms inverted fir tree/pine tree appearance gelatin/ tube media 4. non motile 5. gamma Lecithinase +, media to detect lecithinase production _______________________________________________ egg yolk agar/ Mc Clung egg media SELECTIVE MEDIA result: (+) opaque zone around the colonies a. B. anthracis Causes ANTHRAX (+) black eschar - most common but least severe -PLET (polymyxin lysozyme EDTA 1- Cutaneous _____________________________________________________________________________ - jobs expose to sheep thallons acetate inhalation of spores - 5% SBA (alternative) 2- Pulmonary / Woolsorter’s disease ___________________________________________________________ - woolsorter's disease, ragpicker's, hide porter's b. B. cereus - MEYP (mannitol egg yolk polymyxin 3- Intestinal anthrax _______________________________________________________________________ - ingestion of affected meat - least common but most severe B agar 4- Injectional anthrax – associated with use of drugs of abuse like heroine - PEMBA (polymyxin egg yolk mannitol bromthymol blue agar Virulence Factor virulence: toxins (B. cereus) - heat or ethanol shock test treatment = a) ________________________________ polypeptide-D-glutamic acid capsule, a. Emetic - vomiting enhance spore formation b) Exotoxin with 3 components: edema factor, lethal factor, protective antigen- function of exotoxin _______________ - cereulide toxin b. Diarrheal Bacillus cereussuspected food (spx) Both are _________________________________ motile and ________________________________________ beta - NHE (non hemolytic enterotoxin - CYTK (cytotoxin K) Bacillus subtilis B. cereus can cause ___________________________________________________________________ food poisoning associated to contaminated fried rice - HBL (hemolysin L) B. subtilis is classified under BSL __________________, 1 (lab contaminant) a.ka. HAY BACILLUS GENUS Clostridium perfirngens a.k.a. ____________________________________; gas gangrene bacillus On BAP ______________________; double/ target hemolysis Lecithinase test ______________ (+) opaque zone around the CAUSES: colonies CLOSTRIDIUM (A) myonecrosis - accumulate in wounds Reverse CAMP Test __________________________________________________________________________ (+) arrow head beta hemolysis VIRULENCE FACTOR B: beta & theta strict anaerobes (B) pigbel dse. - type C food poisoning ___________________________________________________________________________________________ A: alpha & lecithinase catalase (-) BOX CAR morphology (+) result: enhanced hemolysis as shown by arrow head zone of beta hemolysis Clostridium tetani Lollipop Bacillus, Tack head Bacillus, tennis racket bacillus, Drumstick bacillus HISTOTOXIC: C. perfirngens iron loving which causes tetanus NEUROTOXIC: (A) C. tetani TETANIC TRIAD: (A) trismus Can produce TETANOSPASMIN _______________________________________________________________ (virulence factor) -neurotoxin causing paralysis: (A) Spastic paralysis (limited movement) > lock jaw > Risus sardonicus (B) C. botulinum (B) Risus sardonicus ENTEROTOXIC: C. difficile (C) Opisthotonos Diagnosis is often made by ____________________________________________________________________ symptoms, terminal swollen spores (B) Flaccid paralysis (unresponsive) Clostridium difficile Causes Pseudomembranous colitis or _____________________________________________________________________ antibiotic associated diarrhea normal flora Can produce Toxins A & B _______________________________________________________________________________ (virulence factor) Toxin A - enterotoxin; Toxi