MTAP 2 Bacteriology PDF, 04-01-23

MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 BASIC INFORMATION BACTERIAL CELL STRUCTURE: GENERAL CHARACTERISTICS OF BACTERIA...

MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 BASIC INFORMATION BACTERIAL CELL STRUCTURE: GENERAL CHARACTERISTICS OF BACTERIA CELL WALL 1. Prokaryote - bacteria have NO true nucleus MANY bacteria have cell wall 2. With both RNA & DNA - compared with viruses, Also known as murein layer or peptidoglycan they have BOTH RNA and DNA; viruses have layer EITHER RNA or DNA Main component is peptidoglycan 3. Usually/majority with CELL WALL except Genera Give shape to the organism Mycoplasma and Ureaplasma → cell wall ○ Round shaped organisms are called deficient cocci 4. SOME can produce spores - take note: NOT ALL ○ Rod shaped organisms are called are capable of producing spores; Genus bacilli Bacillus and Clostridium → spore forming ○ Spiral TOXINS Provides attachment/anchorage for flagellum SOME bacteria produce toxins (organ for locomotion) The ability to produce toxins can be a Usual site/target of antibiotic action virulence factor ○ Antimicrobials are developed to kill Many bacteria are pathogenic and one bacteria and one way to kill organisms reason for that is because they are capable of is by damaging the cell wall producing toxins ○ Cell wall is the primary target in the Take note: toxins are BIOLOGICALLY development of antibiotics. PRODUCED POISONS Basis of gram staining 2 types of toxins: Exotoxins and Endotoxins ○ In classifying gram (+) and gram (-) 1. EXOTOXINS Disaccharides: N-acetyl-glucosamine & N- Produced by gram (+) except Listeria (gram acetyl-muramic acid positive rod) but take note that the production Gram (+) → with teichoic acid, thicker of exotoxins is not exclusive for gram (+) peptidoglycan layer o Some gram (-) may also produce this but Gram (-) → no teichoic acid, thinner most of the time, it is produced by gram peptidoglycan layer, with outer membrane, LPS (+) (lipopolysaccharide) o Listeria (gram positive rod) → unable to CAPSULE produce exotoxins This is not common to all bacteria. There are only Exotoxins are excreted/released by living some that have it. bacteria NOT all are encapsulated Protein in nature; unstable to heating Usually, gram negative organisms are the ones Examples: that are encapsulated (but not all) ○ TSST 1 (Toxic Shock Syndrome Toxin The capsule is anti phagocytic (prevents produced by Staphylococcus aureus) phagocytosis acting as a virulence factor) ○ Diphtheria toxin (virulence factor Being encapsulated contributes to produced by Corynebacterium pathogenicity diphtheriae) Those with capsule are said to develop colonies ○ Botulinum toxin (virulence factor that are mucoid produced by Clostridium botulinum) Neufeld quellung (capsular swelling test) - done ○ Enzyme coagulase (virulence factor to determine if organism is encapsulated produced by Staphylococcus aureus) Positive for this test = has capsule Staining can also be done to detect capsule 2. ENDOTOXINS Streptococcus pneumoniae, Klebsiella Usually produced by gram negative organisms pneumoniae, Neisseria meningitidis = with Endotoxins are released only after cell death polysaccharide capsule or lysis (recall: exotoxins, on the other hand, Bacillus anthracis = capsule with polypeptide D are excreted/released by living bacteria) glutamic acid LPS or lipopolysaccharide in nature (recall: Pasteurella multocida = capsule with hyaluronic exotoxins, on the other hand, are protein in acid nature) Haemophilus influenzae = capsule with Examples: polyribosyl-ribitol-phosphate ○ Shigella (gram negative rod) → known Pseudomonas aeruginosa = alginate capsule to cause bacillary dysentery or shigellosis (bloody diarrhea) black market 1 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Glycocalyx - less organized compared with Twitching motility Kingella kingae capsule, slimy layer, polysaccharide containing i.e., S. mutans Gliding/sliding motility Capnocytophaga PILI gingivalis singular: Pilus AKA Fimbriae Darting motility Campylobacter spp. Usually found in gram negative organisms (N. gonorrhea) Shooting star motility Vibrio cholerae Two types: Common Pili and Sex Pili Common pili - used for attachment to most cells Corkscrew motility Spirochetes (first step in establishing infection) Chilomastix mesnili Sex pili - for gene conjugation, transfer of (parasite) genetic material (cannot be a virulence) Mechanisms of gene transfer: transduction, INCLUSION BODIES conjugation Represents stored food ENDOSPORES OR SPORES Metachromatic granules- BABES ERNST BODIES- Target of sterilization, contains dipicolinic Volutin seen in Corynebacterium diphtheriae acid/calcium dipicolinate MUCH granules Mycobacterium tuberculosis resistant structures which enables organism to Halberstaedter Prowazek -glycogen containing withstand injurious conditions seen in Chlamydia trachomatis there are only two genera who produce spores: OTHER STRUCTURES Bacillus and Clostridium CELL MEMBRANE – phospholipid bilayer, No STEROLS Protective factor except in Mycoplasma Spores can be round, oval, central, terminal, NUCLEOID – contains the genome and subterminal RIBOSOME – mediates protein synthesis Destroy spores by autoclaving With terminal swollen spores: Clostridium tetani Central spores: B. anthracis SPECIMENS, SPECIMEN COLLECTION AND HANDLING Subterminal spores: C. botulinum IMPORTANT RULES Target of antibiotic activity: cell wall FLAGELLA When to collect specimen (for culture) – during the Singular: Flagellum acute phase of infection (within 72 hours of Organ for locomotion infection) Most cocci are Non-motile o Do not collect after antibiotic therapy Bacilli and Spiral organisms are usually motile Observe aseptic technique, collection containers In spiral organisms (spirochetes), its called Axial must be properly labeled & must be sterile filaments or periplasmic flagella Adequate amount must be collected. True motility - due to presence of flagella Collected samples must be transported without Pseudo motility - “Brownian movement” delay, usually within 30 minutes movement of non motile organisms caused by In the processing of samples, observe LEVEL of the movement of molecules surrounding the PRIORITIZATION organism PRIORITIZATION OF SPECIMENS Methods to detect motility: 1 Critical/ Invasive CSF, amniotic fluid, ○ Hanging drop: presumptive test for pericardial fluid, heart Listeria valves ○ Use of flagellar stains: Grays, Leifson 2 UNPRESERVED Feces, sputum wound ○ Use of semi-solid media like SIM media drainage, urine (without (inoculate by stabbing the media and boric acid) then incubate and then read growth 3 Quantitation Catheter tip, urine, after incubation) required tissues for quantitation Non-motile/negative: if growth 4 Preserved Urine (with boric acid), at the line of inoculation feces, swab in holding Motile/positive: if growth is media outside the line of inoculation 5 Batch Processing Sputum, AFB culture Motility is best observed at Room temperature CSF – first to be processed Tumbling motility @RT Listeria monocytogenes Unpreserved – not placed in transport media, without preservatives black market 2 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Quantitation required - colony counting of culture CSF → Centrifuge → Supernatant is removed → and sensitivity; delayed processing might decrease Sediment → Smear preparation and culture the count BLOOD Purpose of blood culture – requested to rule out bacteremia and sepsis Blood pathogens – CoNS (Coagulase Negative Staphylococci), E. coli, P. aeruginosa, Bacteroides fragilis (classified as an anaerobe) Biomarker for fungemia: C. albicans Potential biomarker for sepsis: Procalcitonin, CRP Blood for culture should not contain contaminants and normal flora URINE Phlebotomy site should be cleansed with 70-95% alcohol → iodine scrub → alcohol rinse Specimen of choice for bacterial culture: Clean Common contaminants: S. epidermidis, Viridans catch midstream streptococci, C. acnes Catheterized for those unable to void; SUPRAPUBIC Dilution of blood to medium – 1:10 (blood broth URINE → Anaerobic culture ratio) Usual request is Culture & Sensitivity to rule out UTI Anticoagulant: 0.025% SPS (sodium polyanethol #1 cause of UTI: E. coli; UTI in young female S. sulfonate) saprophyticus o SPS may be inhibitory to some organisms Other causes of UTI: Klebsiella, E. faecalis such as Neisseria, G. vaginalis, Perform colony count using Calibrated loop to Streptobacillus moniliformis, and determine # of colonies/ml of urine Peptostreptococcus anaerobis Considered UTI is colony count of ________ o Counteract: add 1% gelatin to neutralize # colonies counted x dilution factor = colony the effect of SPS count /ml of urine Not to be used for bacteriologic culture: EDTA Dilution factor if 1ul loop was used x 1000, if 10ul Anticoagulant for viral culture but inhibitory to gram loop was used x100 positive organisms and yeast: Heparin Urine sediment→Incubate→Colony count & Routine blood culture bottles are held for 7 days calibrated loop (Nutrient agar is MHA) o Observe tube for sign of growth (hemolysis, Urine → centrifuge → supernatant removed → bubbles, etc.) sediment→ Smear preparation and culture For Brucellosis detection: 3-4 weeks; For leptospirosis Significant : >100,000 = UTI detection: 8 weeks SPUTUM o There are rapid tests for both RESPIRATORY, M.TUBERCULOSIS, NON-STERILE CSF SAMPLE Specimen that is considered to be the First priority Evaluate quality, Do Bartlett’s classification SPUTUM Specimen collected through Lumbar puncture vs SALIVA (Invasive procedure) If more than 10 SECs and less than 25 PMNs were Collect in 3 tubes: Tube 2 for Microbiology noted : saliva For M.tuberculosis identification, we do Decontamination(to remove contaminants and normal flora) and Digestion (liquefy sample: free trap organism) Gold standard for digestion & decontamination: N-acetyl-L-cysteine (NALC) & Sodium hydroxide (NaOH) Other agents:  Z-TSP (zephiran & Trisodium phosphate)  4% NaOH  Cetylpyridium chloride-Sodium chloride Storage temperature 37C method transport temperature Room temp  5% oxalic acid (spx: w/ P.aeruginosa) For smear preparation & culture use SEDIMENT M. tuberculosis is classified as a BSL III pathogen and therefore culture must be done using BSC class II black market 3 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 THROAT SWAB STAINING OF BACTERIAL CELLS Identification of Streptococcus pyogenes which is Direct/Simple staining considered as the major throat pathogen. o Use of only 1 dye, the color of the dye is the Normal throat flora: Viridans streptococcus resulting color, i.e. methylene blue Always associated with pharyngitis and #1 agent o Differentiation of organism from another is involved is S. pyogenes not possible NASOPHARYNGEAL SWAB o The morphology and arrangement of the Nasopharyngeal swab may be required to detect bacterial cells can be identified using this carrier state of N. meningitidis and also method, i.e., clusters, chains identification of B. pertussis & H. influenzae. Indirect/Negative/Relief staining o Bordetella is the agent of Whooping cough o Only the background and not the organism o If you want to detect H. influenzae, use is stained i.e. india ink nasopharyngeal swab o Organism appears colorless o N. meningitidis is not always a pathogen, it SPECIAL STAINING is also a part of the normal flora. It can be normally detected in the body, and its Used to demonstrate special features of the cell natural habitat is in the nasopharynx or Example: staining of capsule, spores, or flagella oropharynx. Schaffer and Fulton o N. meningitidis can cause meningitis. o Used for staining spores ▪ To detect N. meningitidis in cases in o Primary dye: Malachite Green meningitis, the specimen to be o Counterstain: Safranin collected is CSF o Spores will appear green; other structures ▪ For the detection of N. meningitidis will appear red in its carrier state, use Capsular stains Hiss, Anthony’s, Tyler, Muir nasopharyngeal swab as specimen Spore stain Dorner’s, Schaeffer and For bacterial culture, use Dacron, Calcium alginate. Fulton, Wirtz and Conklin Toxic to Neisseria: Cotton swab Flagella Grays, Fisher and Conn, For viral culture, Dacron, Cotton, Rayon fibers. Toxic Leifson to viruses: Calcium alginate – can inhibit viral Metachromatic Albert’s, Neisser, Ljubinsky, replication granules Ponder, Methylene Blue, STOOL Lindergran, Burke’s technique When receiving stool specimen, Family Polar bodies Wayson stain, Methylene Enterobacteriaceae comes to mind. Blue Detection of GIT pathogens like Salmonella, Spirochetes Levatidi’s Shigella Maybe collected if stool collection is not possible: DIFFERENTIAL STAINING Use rectal swab To differentiate one organism from another Usual media used are EMB, MacConkey, SSA Examples: Gram stain & Acid Fast Bacilli – most Stool specimens for bacterial culture are inoculated common differential staining directly to media. o Gram staining differentiates gram positive # of quadrants streaked on plated media: 4 to gram negative organisms. quadrants o Acid Fast Bacilli staining helps distinguish Gram staining is not usually done acid-fast bacilli from non-acid fast bacilli organisms PURPOSES OF TESTING SPECIMEN Prior to staining, we must make a bacterial smear Blood culture: to rule out bacteremia or sepsis CSF processing: To detect cases of meningitis. GRAM STAINING (G/S) Sputum processing: To detect Mycobacterium Stool processing: To detect stool or GIT pathogens, GRAM GRAM PURPOSE REAGENT especially those under the Family POSITIVE NEGATIVE Enterobacteriaceae Performing urine culture: to rule out repeated UTI Primary Crystal Throat swabs: to detect pharyngitis states due to S. Stain/ Initial Violet Violet Violet pyogenes stain Nasopharyngeal swabs: to detect Bordetella, H. influenzae and carrier state of N. meningitidis black market 4  MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Gram’s ACID FAST STAINING Iodine Mordant (Alkaline) Violet Violet Ziehl Auramine- (not a Neelsen/ Kinyoun’s/ Rhodamine PURPOSE dye) Hot Cold Method (Fluorochrom Acetone Method e) Alcohol (95% Best Best to Decolorizer Ethanol) Violet Colorless meth demonstr Most Alcohol- od for ate AFB in Sensitive Acetone SPUTU Tissues Mixture M Counterstain Auramine/ Primary Carbol Fuchsin Secondary Safranin Violet Red Rhodamine Stain Heat/ Wetting (No GRAM POSITVE: Violet/ Purple Mordant Steaming Agent: Mordant) GRAM NEGATIVE: Red process TERGITOL ***In between the reagent we need to wash with tap Decoloriz 3% Acid Alcohol (HCl + 0.5% Acid water. er Ethanol) Alcohol 0.5% KMnO4 GRAM STAINING Counterst (Potassium Most Critical step in gram staining is ain Methylene Blue Permangana DECOLORIZATION. te) Hucker’s modification – Gram Stain for FUNGI Yellow (Uses Ammonium Oxalate + Crystal Violet) against Carbol fuchsin can be used as counterstain in place Black of Safranin. This may be done to improve staining of Background some gram-negative organisms that are poorly Acid Fast: AFO appear observed stained with Safranin. Red against Blue under LPO RULES IN GRAM STAINING Result Background observed All COCCI are gram (+) except: under OIO **Smears are o Neisseria Non-Acid Fast: Blue easier to o Branhamella read o Veilonella. because of All BACILLI are gram (–) except: better o Mycobacterium background. o Corynebacterium May be o Clostridium used as o Bacillus substitute o Erysipelothrix for o Lactobacillus Malachite Green Methylen o Listeria e Blue Higher forms of organisms like Actinomyces, Counterst Nocardia, Streptomyces, Yeast and Molds are gram ain (+). ***AFB smear size 2×3 cm. All Spiral organisms are reported as Gram (–). Not GRAM stained are: ACID FAST STAINING o Rickettsia To distinguish Acid Fast from Non-Acid-Fast o Chlamydia (intracellular) organisms o Mycoplasma; ACID FAST ORGANISMS – are organisms that are o Ureaplasma (wall less) very hard to stain but once stained they are o Spirochetes (can’t resolved by bright field) difficult to decolorize due to MYCOLIC ACID / HYDROXYMETHOXY ACID that envelopes the bacteria. RULES IN ACID FAST STAINING Rule: All bacteria are Non-Acid Fast except: MYCOBACTERIUM black market 5 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Slightly acid fast is NOCARDIA It contains inhibitor e.g., CTBA (Cystine Tellurite Blood Agar) contains potassium tellurite which serves as an inhibitor CULTURE AND CULTURE MEDIA Culture Any medium that contains all what is 5. Differential Media Media needed to support bacterial growth Used to differentiate organisms that are Growth of organism in culture i.e Pure growing together culture, mixed culture, stock culture E.g. Culture o EMB (Eosin Methylene Blue) and MAC – (used in research i.e., American Type Culture Collection) differentiates lactose fermenters and non-lactose fermenters. MEDIA PREPARATION o TCBS (Thiosulfate-citrate-bile salts- TUBE MEDIA PLATED MEDIA sucrose) – differentiates sucrose fermenters and non-sucrose Weigh Weigh fermenters. Dissolve Dissolve Dispense Sterilize 6. Selective and Differential Sterilize Dispense E.g. o EMB & MAC – gram-positive organisms TYPES OF MEDIA cannot grow ACCORDING TO CONSISTENCY Solidifying Examples 7. Transport Media Agent/Agar Purpose: To maintain viability during 1. LIQUID TSB specimen transport Alkaline E.g., JEMBEC, Cary Blair (Vibrio spp.), 0% Transgrow (Neisseria), Amies Peptone Water BHI 2. SEMI-SOLID 0.5 – 1% SIM 8. Biochemical Test Media 3. SOLID Liquefiable: Used for biochemical testing which aids in EMB, MSA bacterial identification 2 – 3% E.g. Non-liquefiable: Rice medium o Simon Citrate – used for citrate 4. BIPHASIC HBT utilization test o Lysine Iron Agar (LIA) – decarboxylate or deaminate lysine ACCORDING TO PURPOSE o Urea agar – used for urease test 1. General Purpose Contains only what is needed to support 9. Media for Susceptibility Test bacterial growth Mueller-Hinton Agar (MHA Broth and agar No added supplement 10. Motility Test Medium SIM (Sulfur, Indole, Motility) 2. Enrichment Media To enhance bacterial growth BACTERIAL IDENTIFICATION METHODS ↑ bacterial yield Selenite, tetrathionate broth (gram- MANUAL Not reliable negative), alkaline peptone (Vibrio spp.) Prone to human error SEMI- No media prep; inoculate only 3. Enriched Media AUTOMATED With dehydrated substrates Allows the growth of fastidious organisms like i.e API- Analytical Profile Index – Haemophilus spp. uses plastic strips & microtubes It contains blood. with biochemical substrates o the biochemical substrates 4. Selective Media are inoculated with pure Used to promote growth of a particular culture suspension organism and at the same time preventing API 20E: Enterobacteriaceae growth of other organisms API 20A: Anaerobes black market 6 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 More rapid DNASE TEST BBL crystal – one-step; no need for Not definitive for S. aureus the addition of rgts May be carried out to detect the following organisms: SMS AUTOMATED VITEK – bacterial ID and o S. aureus susceptibility o M. catarrhalis VITEK2 o Serratia o uses rgt cards: chromogenic 2 methods: substrates o HCl precipitation method – uses DNase agar + o measures changes in 0.1 N HCl absorbance ▪ (+) result: Clearing of agar around the MALDITOF – more rapid than VITEK colonies o Dye method – involves incorporation of dyes to medium, methyl green or toluidine blue SIGNIFICANT GRAM POSITIVE COCCI AND GRAM ▪ (+) result with methyl green: Clear zone NEGATIVE COCCI around colonies IDENTIFICATION TESTS FOR STAPHYLOCOCCI & ▪ (+) result with toluidine blue: Pink zone MICROCOCCI around colonies CATALASE TEST CEPHALOSPORINASE TEST OR BETA LACTAMASE TEST To differentiate Staphylococci and Micrococci from Purpose: An enzyme produced by S. aureus; Ability Streptococci (coagulase negative) to produce beta lactamase = S. aureus is resistant to Reagent: 3% hydrogen peroxide (H2O2) penicillin or all beta lactams (+) result: Vigorous bubbling Uses: Cefinase disc (commercial disc) with Use of colonies from BAP: false positive Nitrocefin MODIFIED OXIDASE TEST / MICRODASE (+) result: Pink to red color For identification of Micrococcus (rapid detection) NOVOBIOCIN DISK TEST Uses 6% oxidase reagent or tetramethyl-p- phenylenediamine dihydrochloride in Differential test for S. saprophyticus and S. dimethylsulfoxide (DMSO) epidermidis (CoNS) = (-) Mannitol and DNase Use of filter paper or commercial disc S. saprophyticus: Resistant (+) result: Blue color S. epidermidis: Sensitive COAGULASE TEST Staphylococcus – Micrococcus – Definitive test for S. aureus facultative strict aerobe To differentiate the pathogenic S. aureus from other anaerobe Staphylococci Aerobic Methods: + + growth o Slide method – 1st step; detect bound Anaerobic coagulase or clumping factor + – growth o Tube method – proceed if (-) slide; detect free Lysostaphin coagulase S R susceptibility ▪ Require incubation at 37℃ for 4 hrs; if (-) Modified incubate further at RT for 16 hrs – + oxidase test Uses Rabbit’s plasma collected using EDTA Use of citrated plasma: false positive Microdase disk – + (+) result: Clump formation Bacitracin MANNITOL FERMENTATION TESTS R S susceptibility Detect acid production Furazolidone / May be carried out to detect S. aureus which is (+) Furoxone S R Required media: Mannitol Salt Agar (MSA) susceptibility Inhibitor: 7.5% NaCl pH indicator: Phenol red Catalase test + + (+) result: Development of yellow halo around the colonies Benzidine test – + A (+) result would mean that: (1) organism was able to ferment mannitol; and (2) the organism was able to tolerate high salt concentration black market 7  MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Glucose Fermenter Oxidizer PYR Test: o detection of enzyme utilization / OF (produce acid w/ (produce acid pyroglutamylaminopeptidase or medium or w/o air) w/ air) pyrrolidonylamylamidase Hemolysis on S. aureus – beta Gamma o Uses p-dimethylaminocinnamaldehyde BAP hemolytic hemolytic o (+) result: bright red color Coagulase Negative Staphylococci (CoNS) Group B Beta Hemolytic S. (S. agalactiae) saprophytic CAMP Test (Christie Atkins Munch Peterson) S. us o Inoculate known and unknown organisms S. aureus epidermi PYR (-); perpendicular to each other (on BAP), dis Glucosidas then incubate e (+) o Uses BAP as media Yellow ▪ known organism: S. aureus (B- colonies hemolytic) White White Colony (has ▪ unknown organism: S. agalactiae colonies colonies staphyloxanth (CAMP test (+)) in) o (+) result: enhanced arrowhead zone of Catalase beta hemolysis + + + test o (-) result: no enhanced hemolysis Coagulase Hippurate Hydrolysis Test + – – test o Detection of the enzyme Hippurate Mannitol hydrolase or Hippuricase fermentati + – – ▪ Hippuricase: produced by S. on agalactiae Hemolysis o In this test. Sodium Hippurate is hydrolyzed Beta Gamma Gamma on BAP into benzoic acid & glycine. Novobioci ▪ To detect benzoic acid = use n S (> ferric chloride S R (< 16 mm) susceptibili 16mm) ▪ To detect glycine = use ninhydrin ty (5 ug) (rapid) DNase test + – – o (+) result: purple * Novobiocin susceptibility – differentials tests for CoNS Bacitracin aka “Taxo A” For Alpha hemolytic Streptococci IDENTIFICATION TESTS FOR STREPTOCOCCI Streptococcus Pneumoniae Streptococci: Viridans Inoculate on BAP if catalase and LAP (+) Neufeld Quellung (+) S. pneumoniae, Steps: Capsular swelling, most useful to differentiate a. Inoculate on BAP the two b. Determine hemolysis pattern c. Subject to tests Bile Solubility test LAP TEST Required media: BAP (Leucine Amino Peptidase) Reagent : Sodium deoxy chocolate Test for catalase (-), gram (+) cocci (+) result: Lysis of colonies Substrate: Leucine-beta-naphthylamide (-) result: Intact colonies (commercial disk) Reagent: cinnamaldehyde Can be done using broth: (+) result: red color (color development) (+) result is clearing on media; (-) result: no color or slight yellow (-) result is turbidity or partial clearing If LAP (+) & Catalase (-) = possible Strep. o Then detect hemolysis pattern when Optochin Disk Test inoculated in BAP uses TAXO P or ethylhydrocupreine For Group A Beta Hemolytic hydrochloride ( TAXO B – bacitracin) (S. pyogenes) S, pneumoniae: Sensitive Viridans Streptococci: Resistant Bacitracin Disk test: o Susceptible: S. pyogenes o Resistant: S. agalactiae black market 8 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 For Gamma Hemolytic Streptococci - Considered as MARKER OF Group D Non-Enterococci VIRULENCE (major virulence Enterococci factor) Aside from inoculating on BAP and waiting for gamma STAPHYLOKINA - Promotes fibrinolysis, causes hemolytic pattern, perform screening SE dissolution of clot Screening Test: Bile Esculin Hydrolysis Test DNAse/ - Decrease viscosity of exudates (different from bile solubility) THERMONUCLE allowing more mobility Media : BEA ( Bile Esculin Agar) ASE Tube media dispensed as slant BETA - Causes beta hemolysis Indicator : FeCl3 (Ferric ammonium citrate) (+) HEMOLYSIN (+): result brown or black precipitate / blackening Sphingomyelin Differential Tests: PYR, Penicillin Disk Test, Salt Tolerance ase C test -6.5% (+) result in Salt Tolerance test : Turbidity or Growth Hot/Cold Lysin Protein A - Interferes with phagocytosis PYR SAL PENICILLIN ENTEROTOXINS - Food poisoning TEST TOLERANCE - A AND B 6.5% NaCl TSST/TOXIC - Causes Toxin Shock Syndrome Group D (-) No growth (-) S SHOCK Non- SYNDROME Enterococci TOXIN Enterococci (+) Growth (+) R Or Staphylococcus aureus (a normal flora) PYROGENIC EXOTOXIN C Characteristics o Gm (+) cocci in GRAPE LIKE CLUSTERS, medium-sized, butyrous ENTEROTOXIN F colonies (oily looking/buttery EXFOLIATIN - Causes skin desquamation in looking colonies) Scalded Skin Syndrome/Ritter’s Disease o Catalase (+), coagulase-mannitol fermentation-DNAse test (+), PYR (- PVL-Panton - Destruction of WBC ) Valentine LEUKOCODIN o BETA HEMOLYTIC on BAP CATALASE - Catalyze decomposition of Diseases o Toxin Mediated: Food poisoning, hydrogen peroxide in water and Toxic Shock Syndrome, Scalded S. aureus oxygen (causes bubbling) skin syndrome or Ritter Disease produces - Not considered a virulence factor catalase o Non-Toxin Mediated: Boils, Additional: S. epidermidis (normal skin flora); carbuncles, furuncles, cellulitis, causes UTI, stich abscess, causes wound infection, sty (eye Prosthetic Heart Valve Infection infection), impetigo CoNS Virulence Factors Differential test: LIPASE - Initiates skin infection o Novobiocin Susceptibility Test HYALURONIDA - Enhances ability of organism to S. epidermidis SE invade tissues ((Other name: o Normal skin flora DURAN RAYNAL FACTOR) o causes UTI, stitch abscess BETA - Responsible for S. aureus o causes Prosthetic Heart Valve infection LACTAMASE or resistance to penicillin and other o Virulence factor: slime PENICILLINASE beta-lactamase antibiotics production/biotin formation COAGULASE - Causes bacterial cell to ▪ Slime: enhances ability of agglutinate in plasma organism to attach to plastic - Converts fibrinogen into fibrin catheters S. saprophyticus black market 9 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 o Most common cause of: UTI in young Streptokinase Dissolution of clot women, uncertain virulence factor Hyaluronidase Spreading factor o Causes pyelonephritis and cystitis in Erythrogenic or Responsible for rashes in scarlet those with indwelling catheters Pyrogenic Toxin fever MRSA/ORSA STREPTOLYSIN: Beta-hemolytic on BAP Strain of S. aureus resistant to Methicillin, Streptolysin O Oxygen labile, Antigenic Nafcillin, and Oxacillin. Can cause sub-surface o S. aureus: produce beta-lactamase Production hemolysis on BAP which is resistant to beta-lactam of ASO Can cause hemolysis only antibiotics when incubated Resistance of Staphylococcus to penincillinase resistant penicillin is due to PBP2a (Penicillin Streptolysin S Oxygen-stable, non-antigenic binding protein 2 (in cell wall) encoded by Can cause surface hemolysis mecA gene) on BAP Treatment of MRSA: Vancomycin, a Can cause hemolysis when “glycopeptide” = VRSA incubated aerobically Detection methods: 1. Use of Chromogenic agar Group B Streptococci MRSA: Rose to Mauve colony color Non-MRSA: Blue Normal flora Normal flora of female genital tract or colony/colorless/no growth lower GIT; causes Septicemia. 2. Cefoxitin Disk Diffusion Test: Induces expression of PBP2A Streptococcis agalactiae 3. Gold standard for MRSA detection: PCR # 1 cause of neonatal meningitis (molecular) In adults, it can cause Postpartum endometriosis Smith & Brown Classification Hemolysis on BAP Alpha Group C, F, & G o partial/incomplete hemolysis/greening Normal flora: skin, nasopharynx, GI tract, of agar genital tract Beta o complete hemolysis/ clear zone Group D (aka. S. bovis group) around colonies Non-enterococci Gamma o Isolation in the blood may suggest o no hemolysis/ red colonies "colon cancer" o no change on BAP S. equinus, S. gallolyticus, S. infantarius, S. Lancefield Classification alactolyticus Based on cell wall polysaccharide antigen (common C o S. bovis is NOT a valid species anymore carbohydrate) → S.equinus is the new name Encountered in blood cultures of patients w/ bacteremia, septicemia, & endocarditis. Group A Streptococcus pyogenes Presence of S. gallolyticus subspecies Characteristics: gallolyticus, in blood cultures has HIGH Gram (+) cocci in chains, PYR (+), Bacitracin CORRELATION w/ GIT Carcinoma/colon Susceptible cancer Causes: Viridans Bacterial pharyngitis/Strep throat o #1 cause of subacute endocarditis Pyodermal infection – ERYSIPELAS o major throat flora Scarlet fever – strawberry tongue S.pneumoniae Necrotizing Fascitis – rapidly progressing skin o Causes lumbar pneumonia infection Post streptococcal sequelae: Acute glomerulonephritis Both S. pneumonia & Viridans (Group D) and Rheumatic fever (results from repeated pharyngitis) prpduces alpha-hemolytic pattern. VIRULENCE FACTORS Enterococci M protein Major virulence of S. pyogenes; prevents phagocytosis E. faecalis; E. faecium; E. durans; E. avium Protein F Promotes attachment of epithelial Causes UTI & wound infections cells black market 10 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Virulence factors: ability to grow in extreme urealyticum conditions, Adhesins, gelatinase, Cytolosin Same as New York GC-LECT VCA -T plus City but with medium Lincomycin Lincomycin - to further GENUS NEISSERIA inhibit gram (+) Kidney or bean-shaped dicplococci SCREENING TEST Uses Chocolate Agar Plate CAP; No DMSO Requires enriched media CYTOCHROME OXIDASE TEST CHARACTERISTICS ○ Gram (-); Modified Oxidase: Gram (+) = BLUE Gram (-) non-motile, caphophilic color ○ Increased CO2 Reagent: tetramethyl-p-phenylenediamine dihydrochloride (+) Result: PURPLE color. May require transport media - transgrow, JEMBEC TESTS TO DETECT SPECIES Microaerophiles & Capnophiles uses CHO Utilization Test Microaerophilic - candle jar Media: Cystine Trypticase Agar; Indicator: Phenol MEDIA Red SELECTIVE In an acid pH (+) media will turn YELLOW; Alk pH = INHIBITORY RED (-) MEDIA FOR AGENTS ISOLATION RESULTS IN ACID Vancomycin CHO PRODU Thayer inhibits g (+) UTILIZATION CED Martin Colistin inhibits g (- TEST FROM: (enriched VCN ) chocolate Nystatin anti- Glucos Malt Lact Sucr Fruct agar) Specie fungal e ose ose ose ose Same as Thayer N. + - - - - Martin but with gonorrhoeae Trimetophrim - Modified LACTATE N. + + - - - Thayer VCN - T ○ Prevent meningitidis Martin swarming organisms -> N. lactamica Proteus mirabilis ONPG + + + + - - Same as Modified Enterobacter Thayer Martin but iaceae to Martin Lewis VCA - T Nystatin is replaced by detect Anisomycin (anti- LLF/SLF fungal) N. cinerea New York Same as Martin Lewis City but Anisomycin is N. flavescens - - - - - (used for replaced by other Amphotericin B (anti- N. elongata VCA - T urogential fungal) pathogens: N. sicca M. hominis + + - + + and U. N. mucosa black market 11 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 o Catalase (-) N. subflava + + - V V o Beta hemolysis on BAP Normal flora MORAXELLA - - - - - Causative agent of Otitis Media Neisseria gonorrhoeae GRAM POSIITVE BACILLI – SPORE FORMERS Always a PATHOGEN GENUS BACILLUS Pelvic inflammatory disease Primary Virulence Factor: Pili Bacillus Anthracis FERMENTS GLUCOSE Characteristics that will differentiate B. SUPEROXOL TEST (+) Rapid Test for N. gonorrhoeae anthracis from another Bacillus spp: Non-motile Causes: and Gamma hemolytic ○ Gonorrhea STD Forms the so called “disjointed bamboo fishing Opthalmia neonatorum – a gonococcal eye rod appearance” infection acquired by newborns when discharge o Typically has square ends from infected mother accumulates in their Colonies: o Colonies with tenacious (sticky) conjunctiva consistency ○ TREATMENT; CREDE’S PROPHYLAXIS o When lifted using a loop it will stand ✓ Dacron Swab like a beaten egg white ✓ Rayon Swab Colonies may show swirling projections giving X Cotton Swab it a Medusa head or lion head appearance 30% H2O2 (+) bubbling Forms the so-called string of pearl pattern Neisseria meningitidis o Due to susceptibility to penicillin o Colonies with comet tail appearance Primary Virulence factor: Capsule Colonies with cut-glass appearance or frosted Neufeld Quellung Test POSITIVE glass appearance FERMENTS GLUCOSE and MALTOSE Forms inverted fir tree/pine tree appearance in Can be a normal flora, natural habitat is oro and gelatin media (semi solid) nasopharynx. (Nonencapsulated strains) Tests Specimen to detect carrier state Nasopharyngeal o Lecithinase (+) Swab Causes: ▪ Media to detect lecithinase ○ Bacterial Meningitis: 5-29 years old production: Egg yolk agar ○ Meningococcemia - N. meningitidis in the (EYA) blood ▪ (+) result: opaque zone ○ Waterhouse Friderichsen Syndrome – severe around colonies form of meningococcemia characterized by o Gelatin hydrolysis (-) bleeding of adrenal glands/DIC o No growth in PEA agar ○ PID which may cause sterility, perihepatitis or o Ascoli test – serologic test for the Fitz Hugh Curtis Syndrome diagnosis of anthrax To collect specimen use o Causes anthrax ________________________________ Calcium alginate Cutaneou Pulmonary / Intestinal Injectional and cotton swab may be inhibitory to N. gonorrhoea s woolsorter’s anthrax anthrax Moraxella catarrhalis disease I Identification tests (+) Ragpicker’ Associate o Oxidase (+) because of the indophenol Black s disease Ingestion d with use o Fermentation: non-saccrolytic; (-) eschar Hide of of drugs of carbohydrates Least porter’s dse improperl abuse like o Butyrate disk test: rapid test; reagent is severe Inhalation y cooked heroine indoxyl butyrate; (+) blue color that can be Most of spores infected obtained with 5 minutes. common while meat o Dnase test (+) Direct handling Most o Unique colonies: hockey puck colonies that contact wool severe could remain intact when pushed to the side of the agar. black market 12 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Direct Least - C. difficile: Pseudomembranous colitis; contact common antibiotic associated diarrhea CLOSTRIDIUM PERFRINGENS Ingestion Aka: Gas Gangrene Virulence factor Tests: a. D-glutamate capsule (not polysaccharide) 1. On BAP: b. Exotoxin with 3 components: edema factor, - target/ double hemolysis (inner beta; lethal factor, protective antigen outer alpha). o Function of exotoxin: to mediate cellular destruction 2. Lecithinase test: positive (enzyme lecithinase) Selective medium: PLET (Polymyxin Lysozyme EDTA Thallous acetate) 3. Reverse CAMP Test: Bacillus Cereus & Bacillus Subtilis - Known organism: S. agalactiae Both are motile - (+): enhanced hemolysis as shown by BACILLUS CEREUS arrow head zone of beta hemolysis o Fried rice bacillus - Giving it a Box Car morphology o Virulence factor: exotoxin/enterotoxin - inner beta; outer alpha hemolysis cholera like toxin ▪ beta: due due to beta o Causes food poisoning hematoxylin o Produces 2 types of toxin ▪ alpha: due to alpha toxin and o Best specimen for testing: suspected food Lecithinase o (+) gelatin hydrolysis o (+) growth on PEA Causes: BACILLUS SUBTILIS - Gas gangrene (myonecrosis) o Hay bacillus - Type C food poisoning o Classified under BSL 1 - Necrotic Enteritis o Opportunistic pathogen - Pig Bell disease (consuming contaminated o Causes eye infection in heroin addicts pig meat) Blood bank contaminant @ RT CLOSTRODIUM TETANI o Penicillin resistant Assacharolytic o Selective media: Mannitol egg yolk AKA: Lollipop Bacillus, Tack Head Bacillus, Tennis polymyxin B agar Racket Bacillus, Drumstick bacillus Produces: Tetanospasmin o blocks neurotransmitter, causing GENUS CLOSTRIDIUM spastic paralysis Signs of Tetanus: Sporeformer - sporulates anaerobically o Lock jaw (trismus), Risus sardonicus Anaerobic (must be cultivated in a gas pak jar) (Devil’s grin), spastic paralysis Catalase (+) Diagnosis is made by: Habitat: human and animal o Symptoms Saccharolytic except C. tetani and C. septicum o Terminally located swollen spore CLOSTRIDIUM DIFFICILE Saccharolytic: ability to ferment at least 1 sugar. Causes: Pseudomembranous colitis or Antibiotic associated diarrhea 3 types of Clostridium: Produces: Toxins A and B: Neurotoxic o A = enterotoxin - destroys some neurotransmitters which causes o B = cytotoxin paralysis Media to isolate CCFA: Cycloserine Cefoxitin - C. tetani: Tetanus fructose agar: Horse stable/barn yard odor - C. botulinum: Foodborne botulism; infant (+) BAP: develop colonies that Fluoresce botulism; SID (Sudden infant death syndrome) chartreuse (yellow fluoresce) Histotoxic Diagnosis: through Cytotoxin detection using - C. perfringens: Gas gangrene/ myonecrosis; immunoassay Enteritis necroticans CLOSTRIDIUM BOTULINUM Enteric Aka: Canned good Bacillus black market 13 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Causes: Botulism-fatal type of food poisoning o Smallest: Mitis Produces: Botulinum toxin- powerful neurotoxin. o Infant botulism may develop as a result of spore 2 TYPES OF C. DIPHTHERIAE: ingestion via breast feeding, baby will manifest Respiratory – affects tonsils/pharynx that can FLOPPY BABY SYNDROME lead to respiratory obstruction. Spores: subterminally located, spherical Cutaneous – common in tropic TOXIGENECITY TEST GRAM POSITIVE BACILLI – NON-SPORE FORMERS MODIFIED ELEK’S Animal Inoculation / Corynebacterium diphtheriae Guinea Pig Lethal Test AKA Kleb Loeffler’s Bacillus In vitro toxigenicity In vivo Causative agent of diphtheria test Suspension of Non-motile and highly pleomorphic On agar media, isolated strain of Morphologically arrangement place a filter C. diphtheriae o Club shaped appearance - one end paper strip with (0.3 ml) is injected diphtheria anti- subcutaneously typically swollen toxin, streak + into thigh of two o Palaside arrangement – side by side control at right guinea pigs, one arrangement angle to the strip protected o Picket fence arrangement of anti-toxin, intramuscularly streak negative with 500 units of o X, y, v, l formation – chinese letter control in the diphtheria anti- arrangement same manner. toxin 18-24 hours On BAP: Beta-hemolytic Unknown culture before the test. Media for isolation: suspected of C. (+) result: The a) Leoffler’s serum & PAI’s coagulated diphtheriae is unprotected streaked parallel animal dies within egg – will stimulate granule formation to + and - control. 2-3 days with & pleomorphism Incubate at 35 evidence of b) CTBA -cystine tellurite blood agar degC for 24 – 48 hemorrhage in Primary isolation media hrs. the adrenal (+) result: Lines of glands. (+): Gray to black colonies precipitation with Obsolete Selective media arc of identity Inhibirot: Potassium tellurite Obsolete c) Tinsdale medium – black colonies with brown halo Guinea Pig A (Protected) – Inject Anti- Non toxigenic diphtheria toxin Toxigenic C. diphtheriae causes DIPTHERIA – Guinea Pig B (Unprotected) – No anti-toxin low grade fever, mild sore throat & body Both shall receive culture suspension (C. malaise. diphtheria) Virulence Factor: Diptheria toxin (+): death of unprotected guinea pig. Characteristic symptom: Pseudomembrane – SHICK’S TEST BULL’S NECK APPEARANCE o Immunity/susceptibility test for Initial site of infection: Epithelial diphtheria cells of tonsils o Detection if a person is at risk or not If (+) sa diphtheria, initial site is VERO CELL CYTOTOXITY ASSAY – gold standard tonsils. for diphtheria toxin production. (+) growth on media will require detection of DICK TEST - method of determining toxin production susceptibility to scarlet fever - S. pyogenes Appears beaded when stained with methylene blue because of Babes Ernst metachromatic granules ACID FAST BACILLI – GENUS MYCOBACTERIUM Colony types: Gravis, mitis, belfanti, and Aerobic intermedius – based on the phenotypic Non motile characteristics of size, texture, color, hemolysis, Slow growers and requires whole egg for and presence of metachromatic granules growth. o Largest: Gravis black market 14 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 Difficult to stain, once stained it is difficult to avium decolorize due to hydroxy methoxy complex. acid/mycolic acid. BIOCHEMICAL TEST SPECIMEN PROCESSING BIOCHEMICA REQUIRED REAGENT (+) RESULT Digestion and Decontamination L TEST MEDIA 1. Decontamination – remove contaminants NIACIN TEST Lowenstein and normal flora. Jensen NIACIN TEST 2. Digestion – liquefy sample; free any trap AND NITRATE Media Strip organism. REDUCTION impregnate Inoculate in Gold standard for these process: (Positive in d with YELLOW Lowenstein M. CYANOGEN (slant) → 1. N-acetyl-L-cysteine – Decontaminant tuberculosis BROMIDE inoculate → 2. Sodium hydroxide (NaOH) – Digestion and incubated → Other agents: Negative in Yellow color Diff. M. bovis) 1. Z-TSP (zephiran & Trisodium phosphate) After Initial (+) incubation RED (-) no 2. 4% NaOH – both digestion & we add: n-n color decontaminant -dimethyl-l- change 3. Cetylpyridium chloride Sodium chloride naphthylami method ne sulfanilic After NITRATE Sodium 4. 5% oxalic acid – for specimen likely to REDUCTION Nitrate Broth acid adding contain Pseudomonas aeruginosa. zinc CULTURE MEDIA to confirm a powder: (-) result we (+) no Non- Agar Selective Liquid add: zinc color selective based dust/zinc change (-) media powder RED Egg based Clear Antibiotics These are Developm media with media, non- Lowenstein 20% Ferric IRON UPTAKE ent of rusty malachite easy conventio Jensen Ammonium TEST brown Media Citrate green, examinati nal media color inhibits on of ARYLSULFATA contaminati colonies SE TEST ng growth in organisms; 10-12 Detection of Phenolphthal 2N Sodium Pink Color growth in 6- days. rapid ein media carbonate 10 weeks. growers (+) Common disadvantage: Prolonged M. fortuitum- time of growth chelonae Heating of Lowenstein Middlebro Gruft Bactec colonies at Jensen ok 7H10 Modified 12B HEAT STABLE 68 degC Media: with Lowenstei CATALASE prior to the Common dextrose n Jensen Bactec TEST addition of Selective 13A Tween 80 reagent is Petragnani: Middlebro To detect Media required Bubbling More ok 7H11 Middlebro Septi-chek heat labile (Heat for 20 inhibitory; with ok 7H11 catalase mins. → let recommend casein Middlebro Mycobacteri cool before ed for sterile. hydrolysat Mitchison ok 7H9 um like M. adding 30% e Selective tuberculosis hydrogen Dorset Egg 7H11 peroxide) Media Mitchison Useful in the identification of M. kansasii 7H11 TWEEN 80 is the only positive in 3 days Wallenstein HYDROLYSIS M. tuberculosis (+) in 10-20 days Medium: M. TEST (+) result: development of pink color Indicator: Neutral RED black market 15 MTAP 2 BACTERIOLOGY AUTHOR: MA’AM LIWANAG 04-01-23 T2H/ TCH M. leprae susceptibility a.k.a HANSEN’s Bac