Bacteriology Comprehensive Exam Reviewer PDF

BACTERIOLOGY COMPREHENSIVE EXAM REVIEWER — JEM 3E STAINING OBJECTIVES OF STAINING ​ Determine the morphology ​ Differentiate groups of bacteria ​ Identify organisms with special structures BASIC DYES ​ Common...

BACTERIOLOGY COMPREHENSIVE EXAM REVIEWER — JEM 3E STAINING OBJECTIVES OF STAINING ​ Determine the morphology ​ Differentiate groups of bacteria ​ Identify organisms with special structures BASIC DYES ​ Commonly used in bacteriology ​ These are cationic dyes with positively charged groups that adhere to negatively charged molecules. ​ Includes: METHYLENE BLUE, BASIC FUCHSIN, CRYSTAL VIOLET, SAFRANIN, MALACHITE GREEN ACIDIC DYES ​ These are anionic dyes with negatively charged group that bind to positively charged cell structures. ​ Includes: EOSIN, ROSE BENGAL,ACID FUCHSIN SIMPLE STAINING ​ Application of single staining solution ​ Basic dyes have greater affinity for nuclei; Acid dyes have greater affinity for cytoplasm ​ Directed towards the forms and shapes of the cells ​ Example: METHYLENE BLUE, IODINE DIFFERENTIAL STAINING ​ Application of 2 or more stains ​ Makes use of primary stain, mordant, decolorizer and counterstain ​ Example: GRAM STAINING, ACID FAST STAINING NEGATIVE STAINING ​ Background staining, not the organism itself ​ Results the bacteria appearing as light colored bodies against the dark background Examples: ​ INDIA INK = Cryptococcus neoformans (for the brain, fatal) ​ NIGROSIN GRAM STAIN ​ Named after HANS CHRISTIAN GRAM and CARL FRIEDLANDER CARL FRIEDLANDER ​ Discovered Klebsiella pneumoniae SPECIAL STAINS STAIN TECHNIQUE STAIN CELL STRUCTURE Dyar Congo Red Cell Wall Anthony’s, Hiss’, Gin’s Crystal Violet Capsule Nigrosin Nigrosin Dye (Black) Capsule  Neisser Methylene Blue & Crystal Violet Metachromatic granules Albert Malachite Green & Toluidine Blue Metachromatic granules Dorner Carbol fuchsin & Nigrosin Dye Endospore Schaeffer-Fulton Malachite Green and Safranin Red Endospore Gray Carbolfuchsin & Tannic Acid Flagella (Mordant) Leifson Carbolfuchsin & Tannic Acid, MB Flagella Feulgen Carbol Fuchsin DNA Levaditi’s Silver Nitrate Spirochetes Fontana-Tribondeau Ammoniacal Silver Nitrate & Tannic Spirochetes acid PRIMARY STAIN CRYSTAL VIOLET COUNTERSTAIN SAFRANIN GRAM POSITIVE CELL ​ Thick peptidoglycan with numerous teichoic acid cross linkages WALL ​ Plasma membrane has lipoteichoic acid ​ Has penicillin-binding protein = thus, can inhibit GRAM NEGATIVE CELL ​ Thin peptidoglycan WALL ​ Has outer membrane ○​ Periplasmic space → houses peptidoglycan inner membrane ​ More potent because of lipopolysaccharides in outer layers BETA-LACTAMASE = fends off penicillin (not all organisms have this) GRAM STAIN PROCEDURE ​ V = crystal violet → purple/blue color if gram (-) ​ I = iodine (causes crystallization) ​ A = alcohol decolorizer ​ S = safranin → red/pink color if gram (+) 1.​ HEAT FIXATION 2.​ CV IS ABSORBED BY THE PEPTIDOGLYCAN 3.​ IODINE ACTS AS A MORDANT 4.​ DECOLORIZER 5.​ SAFRANIN STAINS BACTERIA PINK (did not retain CV dye) PRECAUTIONS ​ If the crystal violet dye is rinsed too vigorously prior to the application of iodine, it will not be retained and will leave the gram negative bacteria unstained. ​ If the decolorization is prolonged, the Gram – positive complex will be removed and the gram positive bacteria will not be stained. ​ If the decolorization is insufficient, the organism may falsely appear as gram positive cells. ​ If the safranin dye is left applied for more than a minute, the Gram positive complex will be eliminated from the previously stained cells. ​ Failure to leave the safranin dye for a sufficient time will result in unstained gram – negative bacteria and background materials. GENERAL RULE ​ All cocci are gram–positive except for: ○​ Neisseria ○​ Veilonella ○​ Branhamella (Moraxella). ​ All bacilli are gram–negative except for: ○​ Actinomadura ○​ Arcanobacterium ○​ Bacillus ○​ Clostridium ○​ Corynebacterium ○​ Erysipelothrix ○​ Gordonia ○​ Kuthria ○​ Listeria ○​ Mycobacterium ○​ Nocardia ○​ Rhodococcus ○​ Streptomyces ○​ Tropheryma whipplei ○​ Tsukamurella. WHY GRAM (+) BECOMES ​ Removal of MgRNA GRAM (-) ​ Aged, dying, and autolyzing cells ○​ Old cells may lose their ability to retain stains. ○​ Antibiotic – treated bacterial cell have atypical staining reaction. ​ By using acidic iodine during staining ​ Due to technical error or the wrong use of stains EXCEPTIONS IN GRAM ​ CHLAMYDIA = Organisms that exist almost exclusively within host cells STAINING ​ MYCOPLASMA AND UREAPLASMA = Organisms that lack cells walls ​ SPIROCHETES = Organisms with insufficient dimension to be resolved by light microscopes ACID FAST STAINING ​ It is used to stain bacteria that have high lipid contents in their cell walls. ​ It utilizes carbolfuchsin as the primary stain and methylene blue or malachite green as the secondary stain. ​ In this procedure, the cell wall of acid–fast bacteria resists the acid–alcohol (hydrochloric acid – ethanol mixture) decolorization step. ​ Heat is applied as a mordant in the Ziehl Neelsen Method while tergitol is used in the Kinyoun Method MAINLY FOR MYCOBACTERIUM TUBERCULOSIS PROCEDURE OF AFS ​ PRIMARY STAIN = carbolfuchsin ​ DECOLORIZER = acid alcohol (HCL + ETHANOL) ​ SECONDARY STAIN = methylene blue, malachite green The primary stains binds to mycolic acid in the cell walls of acid fast bacteria, and is retained after decolorizing with acid alcohol. Acid Fast Bacilli (AFB) retain the primary stain and deep-pink or red-colored while non – AFB are either blue or green colored. METHODS OF AFS ​ ZIEHL-NEELSEN = Hot staining ​ KINYOUN = Cold staining ​ PAPPENHEIM = differentiates Mycobacterium smegmatis ​ BAUMGARTED = differentiates Mycobacterium leptae ​ AURAMINE-RHODAMINE = fluorescent staining; fluorochrome; selective for the cell wall of AFB. FACILITATING AFS ​ The use of heating or steaming process for five to seven minutes to temporarily remove the mycolic acid, while the smear is flooded with stain. ​ By increasing the concentration of dye and phenol in the staining reagent. ​ A prolonged contact of the specimen with the primary stain. ​ The addition of wetting agent like tergitol. DSSM ​ 3 MORNING FIRST SPUTUM ​ TWO KINDS: ○​ Expectorated = deep cough ○​ Induced = aerosis  BARLETT’S CRITERIA ​ 25 PMNs MEDICINES 1ST LINE (RIPES) 2ND LINE (CCOKE) ​ RIFAMPICIN ​ CIPROFLOXACIN ​ ISONIAZID ​ CAPREOMYCIN ​ PYRAZINAMIDE ​ CYCLOSERINE ​ ETHAMBUTOL ​ OFLOXACIN ​ STREPTOMYCIN ​ KANAMYCIN ​ ETHIONAMIDE MDR-TB (multidrug resistant TB) = RESISTANT TO RIFAMPIN AND ISONIAZID XDR-TB (extra drug resistant TB) = RESISTANT TO 1ST LINE OF DRUGS AND FLUOROQUINOLONES CULTURE MEDIA AST ANTIMICROBIAL ​ performed on bacteria and fungi isolated from clinical specimens to determine which antimicrobial agents might be effective in treating SUSCEPTIBILITY infections caused by these organisms. TESTING MIC ​ susceptibility testing on bacteria can be performed by classic disk diffusion or dilution (minimal inhibitory concentration [MIC]) methods. CLSI ​ Protocols that describe these methods are published and frequently updated by the Clinical and Laboratory Standards Institute (CLSI) in the United States. ​ The European counterpart is The European Committee on Antimicrobial Susceptibility Testing (EUCAST). ​ Worldwide, antimicrobial susceptibility testing is guided by the International Organization for Standardization (ISO). This organization is a worldwide federation of national standards bodies. REASONS FOR ​ Antimicrobial susceptibility testing should be performed on a bacterial isolate from a clinical specimen if the isolate is determined to be a PERFORMING TESTS probable cause of the patient's infection and the susceptibility of the isolate to antimicrobials cannot be reliably predicted based on previous experience with the bacteria at a specific health care facility. ​ Susceptibility testing of isolates can also provide information on decreases in the susceptibility of bacteria to antimicrobials. EPIDEMIOLOGIC ​ A separate set of critical MICs other than the MICs used to judge a bacterium susceptible or not susceptible to an antimicrobial are CUTOFF VALUES (ECVs) developed for this purpose. These values are termed epidemiologic cutoff valves (ECVs). ​ ECV MICs separate bacterial populations into those likely to be wild type (susceptible) versus those that are becoming resistant, either by mutation or acquisition of resistance genes. When the ECV MIC value is achieved, it is an indication that the bacterium is likely developing resistance to the antimicrobial. FACTORS TO CONSIDER ​ Body site from which the bacterium was isolated WHEN DETERMINING ○​ Susceptibility tests are not routinely performed on organisms isolated from an anatomic site for which they are normal inhabitants. SUSCEPTIBILITY ​ Presence of other organisms and quality of the specimen from which the organism was grown TESTING ○​ The isolation of an organism in pure culture is less likely to represent contamination than a mixed culture. ○​ The presence of more than two species at greater than 105 colony-forming units (CFU) per milliliter isolated from urine suggests contamination, and these organisms may not require susceptibility testing; however, a pure culture of E. coli at more than 105 CFU /mL would likely represent true infection and would be tested. ​ Host's status ○​ The host status of the patient often influences susceptibility testing decisions. In an immunocompromised patient, species usually viewed as normal microbiota might be responsible for an infection and therefore may require susceptibility testing.​ SELECTING ​ Antimicrobial package inserts written by the U.S. Food and Drug Administration (FDA) should be consulted for information concerning the ANTIMICROBIAL dosing and indications for which the antimicrobial was approved and the performance of the anti-microbial agent during initial clinical trials. AGENTS FOR TESTING ​ The patient population must be considered in the choice of antimicrobial agents to be tested. Some agents are contraindicated in pediatric patients FLUOROQUINOLONES IMPAIR CARTILAGE DEVELOPMENT IN PEDIATRIC PATIENTS TETRACYCLINE DAMAGE DEVELOPING TEETH IN PEDIATRIC PATIENTS SELECTION OF TEST ​ Generally, a laboratory will define a battery of 10 to 15 antimicrobial agents for routine testing against the BATTERIES ○​ Enterobacterales ○​ Pseudomonas spp., ○​ nonfastidious gram-negative bacilli (e.g., Acinetobacter spp., Stenotrophomonas maltophilia, and Burkholderia cepacia),  ○​ staphylococci, and enterococci. ​ Sometimes a separate battery is performed for urine isolates, representing drugs appropriate for treating urinary tract infections (UTIs). A supplemental battery that contains antimicrobial agents with enhanced activity may be included by laboratories that encounter a significant number of bacteria resistant to the more commonly used antimicrobials. PRIMARY TENANT ​ A primary tenet of anti-microbial therapy is to use the least toxic, most cost-effective, and most clinically effective agents and to refrain from use of costly, broader-spectrum agents. CASCADE REPORTING ​ CLSI provides guidance for development of a selective or so called cascade reporting protocol. ​ As a general guideline, it is suggested that within an antimicrobial class, primary (group A) agents be reported first and secondary (group B) agents be reported only if one of the following conditions exists: ○​ The isolate is resistant to the primary agents. ○​ The patient cannot tolerate the primary agents. ○​ The infection has not responded to the primary agents. ○​ A secondary agent would be a better clinical choice for the infection (e.g., meningitis. ○​ Bacteria were isolated from another site, and a secondary agent might be useful for treating both infections. For example, a primary cephalosporin, such as cefazolin (a first-generation cephalosporin), would be a reasonable choice for a susceptible E. coli, and secondary cephalosporins, such as cefuroxime (second-generation cephalosporin) or cefotaxime (third-generation cephalosporin), would generally not be required. An exception would occur with meningitis because third-generation cephalosporins cross the blood-brain barrier much more effectively than their first-generation counterparts. Gentamicin = aminoglycoside of choice for treating serious infections caused by gentamicin-susceptible Pseudomonas aeruginosa Tobramycin or amikacin = gentamicin-resistant isolates. Aminoglycosides are not effective for treating meningitis because they do not readily cross the blood-brain barrier. Secondary agent may be reported if: ​ if the patient has a polymicrobial infection, and a secondary (but not a primary) agent would more likely be effective against all pathogens present. ​ if the patient has a disseminated infection, and a secondary agent would more likely be effective at all sites. Agents with very broad-spectrum activity or increased potency (group C) may be tested and reported for the reasons listed for secondary agents. ​ In addition, group C agents would be considered for routine testing if a particular institution encounters large numbers of isolates resistant to group A and group B agents. Finally, agents with activity only in the urinary tract should be reported only on isolates from urine; these drugs concentrate in the urine and are clinically ineffective in treating other infections. TRADITIONAL ANTIMICROBIAL SUSCEPTIBILITY METHOD INOCULUM ​ Inocula are prepared by adding cells from four to five isolated colonies of similar colony morphology growing on a noninhibitory agar PREPARATION medium to a broth medium and then allowing them to grow to log phase at 35° C for 2 to 6 hours, until they reach the McFarland 0.5 standard ​ Four to five colonies, rather than a single colony, are selected to minimize the possibility of testing a colony that might have been derived from a susceptible mutant. ​ Inocula can also be prepared by suspending colonies grown overnight on an agar plate directly in broth or saline. This direct inoculum suspension preparation technique is required for staphylococci and for bacteria that grow unpredictably in broth (e.g., fastidious bacteria) but can be used for any organism. Because it does not rely on growth in an inoculum broth, the use of fresh (16- to 24-hour) colonies is imperative. MCFARLAND TURBIDITY ​ The most widely used method of inoculum standardization involves comparing the turbidity of the inoculum preparation with McFarland STANDARD turbidity standards. ​ McFarland standards can be prepared by adding specific volumes of 1% sulfuric acid and 1.175% barium chloride to obtain a barium sulfate solution with a specific optical density. ​ The most used is the McFarland 0.5 standard, which contains 99.5mL of 1% sulfuric acid and 0.5mL of 1.175% barium chloride. ​ Stored in room temperature in a dark room ​ The McFarland 0.5 standard provides turbidity comparable with that of a bacterial suspension containing approximately 1.5 × 108 CFU/mL. INOCULUM ​ If suspension is too dense = dilute with additional sterile broth or saline STANDARDIZATION ​ If suspension is too light = more organisms are added or the suspension is reincubated A convenient and more precise alternative to the traditional, highly subjective visual adjustment to match the McFarland standard is the use of a nephelometer or spectrophotometer. INTERPRETATIONS ​ Susceptible indicates that the bacteria are inhibited by achievable concentrations of the agent in vivo when the recommended dose is administered to treat an infection ​ Intermediate implies that antimicrobial MICs approach the usual blood and tissue levels, and response rates might be lower than for susceptible isolates. This applies to agents with lower toxicity that can be prescribed at higher-than-normal doses. ​ Resistant implies that isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage. ​ Nonsusceptible is used for isolates for which only a susceptible breakpoint is designated because of the absence or rare occurrence of resistant strains. For each antimicrobial agent, the MIC breakpoint separates susceptible from resistant results. Organisms with MICs at or below the breakpoint are  susceptible, and those with MICs above that breakpoint are intermediate or resistant. Broth macrodilution, broth microdilution, and agar dilution methods are used for determining MIC values. ​Growth may be seen as turbidity, a haze, or a pellet in the bottom of the well. Results for quality control (QC) organisms should be read before reading results for patient isolates to determine whether the test was performed correctly. TEMPERATURE ​ Temperature at or below -60° C is optimal and necessary for more temperature-labile drugs such as imipenem and clavulanic acid; ​ -20° C storage is acceptable for some agents. Antimicrobial solutions are not to be refrozen after thawing. BREAKPOINT (CUTOFF) ​ Breakpoint (cutoff) is the term applied to the concentration of an antimicrobial agent that coincides with a susceptible or intermediate MIC breakpoint for a particular drug. When two concentrations are tested and no growth is present in either well, the isolate is susceptible. When there is growth in the low concentration but no growth in the high concentration, the isolate has intermediate susceptibility, and a resistant isolate grows in both wells. The qualitative interpretation nonsusceptible, susceptible, intermediate, or resistant rather than an MIC value is reported. The primary advantage of breakpoint over full MIC testing for a drug is that a greater number of drugs can be tested on a single panel because a smaller number of wells is needed. The primary disadvantage of breakpoint testing is that a precise MIC is not obtained. Standalone breakpoint panels are generally not currently used clinically. TRAILING ​ Trailing involves heavy growth at lower concentrations followed by one or more wells that show greatly reduced growth in the form of a small button or a light haze. ​ This commonly occurs with sulfonamides, trimethoprim, and trimethoprim-sulfamethoxazole; the mode of action of the agents allows the bacterial cells to grow through several generations before inhibition. In this case the trailing is ignored, and the end point is read as an 80% reduction in growth compared with the growth control. Trailing with most other drugs may represent contamination and should not be ignored unless it is known that trailing commonly occurs with the antimicrobial agent-organism combination. One shortcoming of the broth microdilution MIC method is its inability to produce a penicillin MIC that is consistently within the resistant range for staphylococci that are low-level, B-lactamase producers. AGAR DILUTION ​ The shelf life of agar dilution plates is only 1 week for most antimicrobial agents stored at 2° to 8° C because many drugs are labile in this temperature range. ​ Because plate preparation is laborious and this procedure is practical only if large numbers of isolates are tested, agar dilution is generally performed in research settings, although it is currently considered the primary method for antimicrobial susceptibility testing of obligate anaerobes and is the only accepted reference method for Neisseria gonorrhoeae and Helicobacter pylori. DISK DIFFUSION TESTING DISK DIFFUSION ​ The disk diffusion test, commonly known as the Kirby-Bauer test, has been widely used in clinical laboratories since 1966, when the first TESTING standardized method was described. ​ Briefly, a McFarland 0.5 standardized suspension of bacteria (as described in Inoculum Preparation earlier) in Mueller-Hinton broth is swabbed over the surface of a standardized Mueller-Hinton agar plate, and paper disks containing specific concentrations of antimicrobial agent are placed onto the inoculated surface. After incubation of 16 to 18 hours, the diameters of the zones of inhibition around each disk are measured. The result is interpreted as nonsusceptible, susceptible, intermediate, or resistant to a particular drug according to preset criteria. DISK STORAGE ​ For long-term storage, disks are stored at -20º C or below in a non-frost-free freezer. ​ A working supply of disks can be stored in a refrigerator at 2° to 8° C for at least 1 week. Disks should always be stored in a tightly sealed  container with desiccant. The container should be allowed to warm to room temperature before it is opened to prevent condensation from forming on the disks when warm room air contacts the cold disks. READING PLATES ​ Lawn of growth must be confluent or almost confluent ​ The appearance of individual colonies is unacceptable. END POINT ​ As with dilution tests, the end point for the sulfonamides, trimethoprim, and trimethoprim-sulfamethoxazole is an 80% reduction of growth. Obvious colonies within a clear zone should not be ignored. TRANSMITTED LIGHT ​ Transmitted light rather than reflected light will increase the accuracy of tests with the penicillinase-resistant penicillins linezolid and vancomycin when testing staphylococci and for vancomycin when testing enterococci. BIOTERRORISM ​ Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Brucella spp.  BACTERIOLOGY BACTERIA SUMMARY Jemie 3E (Staph / Strep / Enterococci ) STAPHYLOCOCCUS SPP. S. aureus S. epidermidis S. saprophyticus CATALASE + + + COAGULASE + - - MODIFIED OXIDASE TEST - - - BACITRACIN R R R NOVOBIOCIN S S R DNASE + - - GROWTH ON BAP Medium, convex, creamy, dome shaped white creamy non-hemolytic growth Non-hemolytic on BAP Pigment: white to golden yellow White colonies GROWTH ON CNA + GROWTH ON MSA Acid-producing Can grow but lacks fermentation on MSA MSA fermentation variable (gold yellow) FERMENTATION Mannitol and trehalose fermentation No fermentation Mannitol and trehalose fermentation GROWTH ON CHROMOGENIC AGAR Deep pink to fuchsia colonies Partially or completely inhibited Turquoise VIRULENCE FACTORS - Surface structures - Biofilm production Ability to adhere to epithelial cells of the - Enzymes - cell surface & extracellular compounds urinary tract - Toxins - promote adherence of bacteria to surfaces of prosthetic devices SIGNIFICANCE Cause of infective endocarditis and toxin-induced Cause of hospital-acquired or nosocomial Cause of UTIs predominantly on women and diseases, food poisoning, and is associated with infections catheter assistant UTI scalded skin syndrome (SSS) and toxic shock syndrome (TSS). GRAM POSITIVE GROWS IN 7.5%-10% NaCl CATALASE + LYSOSTAPHIN SENSITIVE NONMOTILE AND NON-SPORE BACITRACIN RESISTANT ALL STAPHYLOCOCCUS SPP. ARE: FORMING FACULTATIVE ANAEROBES MODIFIED OXIDASE - REDUCES NITRATES TO NITRITES  STAPHYLOCOCCUS RESULTS DIFFERENTIATION COMPONENTS / PROCESS / MANUAL TESTS CATALASE TEST (+) bubbles / effervescence (+) Staphylococcus MANUAL: (-) Streptococcus (-) NO bubbles / effervescence 3% hydrogen peroxide COAGULASE TEST (+) gel or clot formation (+) S. aureus Two types of coagulase: BSFT Coagulase reagent (-) S. epidermidis and S. saprophyticus Bound (slide) (rabbit plasma with EDTA (-) NO gel or clot (cons) Free (tube) = BETTER in 0.5 mL amounts) Incubate for 1-4 hours at 35o C to 37o C Can be refrigerated for 10 days or frozen at -20o C for months RAPID DETECTION OF COAGULASE ACTIVITY Latex agglutination Latex particle (coated with human plasma fibrinogen and IgG) → reacts with clumping factor and protein A of SAU MODIFIED OXIDASE (+) dark blue / purple (+) Micrococcus Reagent: TEST (-) NO change in color (-) All Staphylococcus 6% tetramethyl-p-phenylenediamine dihydrochloride in dimethyl sulfoxide Tests if it has cytochrome C enzyme BACITRACIN (+/S) Zones > 10 mm (+/S) Micrococcus and Rothia 0.04 u Bacitracin disk SUSCEPTIBILITY TEST (-/R) Zones < 10 mm (-/R) All Staphylococcus NOVOBIOCIN (+/S) zone > than 16 mm (+/S) other CoNS (S. epidermidis etc.) 5ug of Novobiocin SUSCEPTIBILITY TEST (-/R) zone < or = to 16 mm (-/R) S. saprophyticus DNASE (+) Colorless (Methyl Green) (+) S. aureus For the presumptive identification of S. aureus and 1N hydrochloric acid (+) Bright pink color (Toluidine Blue (-) CoNS differentiates it from CoNS. (DNA + HCl = very fine O) precipitate) (-) No change in color Toluidine Blue O = Bright pink color (detection of thermostable nuclease) Incubate in an inverted position for 24-48 hours at 37o C GERM-TUBE TEST (+) germ tube (appendage that is 1/2 (+) Candida albicans within 2 hours Sabouraud dextrose agar plate (MANUAL) the width and 3-4x length of yeast (-) Candida tropicalis and cell) staphylococci Yeast cells + 0.5 mL of sheep agar (rabbit plasma) and incubate at 35o C for no longer than (-) No appearance 3 hours  STREPTOCOCCI SPP. S. pyogenes (Group A) S. agalactiae (Group B) S. pneumoniae (Group C) CATALASE - - - MODIFIED OXIDASE TEST - - - BACITRACIN S R S SXT R R S OPTOCHIN R R S GROWTH ON BAP colonies are small, translucent, smooth and grayish white mucoid colonies surrounded by a gram positive cocci in pairs that are oval or exhibit well-defined beta hemolysis small zone of B-hemolysis lancet shaped FERMENTATION Ferments carbohydrates Ferments carbohydrates Ferments carbohydrates VIRULENCE FACTORS PROTEIN M group B-specific antigen / capsule Capsule (polysaccharide) INDIGENOUS MICROBIOTA NOT part of indigenous microbiota PART of indigenous microbiota of the female genital PART of indigenous microbiota tract and lower gastrointestinal tract SIGNIFICANCE Pathogenic, acquired through contaminated Nosocomially transmitted by unwashed hands of a Asymptomatic member of normal droplets, highly resistant to drying mother or health care personnel to the newborn or respiratory tract. Cell wall contains C Fever-producing and flesh eating bacterium infant. (Neonatal meningitis and sepsis, SUBSTANCE (Strep throat, scarlet fever) Pneumonia) RUSTY- TINGED SPUTUM Causative agent of LOBAR PNEUMONIAE Gram positive spherical cells that are arranged in chains or pairs CAPNOPHILIC ALL STREPTOCOCCI ARE: (-) catalase, oxidase, gas production Non-motile Facultative anaerobes Culture: appear grayish, pinpoint, and translucent to slightly opaque while some have mucoid colonies All streptococci except the viridans group and S. pneumoniae are included in the Lancefield classification.  CLASSIFICATION OF STREPTOCOCCI ACADEMIC / BERGEY’S CLASSIFICATION SMITH AND BROWN CLASSIFICATION LANCEFIELD CLASSIFICATION Based on TEMPERATURE REQUIREMENT Based on HEMOLYTIC PATTERNS Based on extraction of C CARBOHYDRATE PYOGENIC GROUP VIRIDANS GROUP LACTIC GROUP ENTEROCOCCUS ALPHA HEMOLYTIC BETA HEMOLYTIC GAMMA / LANCEFIELD CLASSIFICATION GROUP STREPTOCOCCI STREPTOCOCCI NON-HEMOLYTIC Neither grow at 10°C Grow at both 45°C It will grow at 10°C and Grow at 10°C, 45°C They have partial / Exhibit a complete lysis Do not exhibit the Based on the extraction of C CARBOHYDRATE nor at 45°C, but only and 37°C 37°C and 37°C incomplete hemolysis of of red blood cells lysis of red blood from streptococcal cell wall at 37°C red blood cells around the cells Not part of the Non-hemolytic and has Part of normal colonies Clear zone of hemolysis Rebecca Lancefield Mostly beta hemolytic Lancefield Group but Lancefield N antigen microbiota of the around the colony The RBC surround reactions some are alpha human intestine For the culture, greenish the colony are hemolytic Often found in dairy E. faecalis or incomplete hemolysis S.pyogenes, unaffected S.pyogenes and products S.agalactiae Groups C and G S. lactis, causes normal S. pneumoniae S. bovis streptococci coagulation or souring of milk STREPTOCOCCUS TESTS RESULTS DIFFERENTIATION COMPONENTS / PROCESS / MANUAL BACITRACIN DISK TEST (S) Susceptibility of any zone of (S) S. pyogenes, S. pneumoniae, Group G Differentiates S. pyogenes from other beta-hemolytic groups and viridans inhibition (TAXO A) (R) S. agalactiae, Viridans streptococci, Group D streptococci, MANUAL: Enterococcus For beta-hemolytic Filter paper impregnated with 0.04 units of bacitracin Lawn technique streap SULFAMETHOXAZOLE AND (R) Exhibits resistance (R) S. pyogenes, S. agalactiae, Enterococcus TRIMETHROPRIM TEST (S) S. pneumoniae, Viridans streptococci BILE ESCULIN AGAR (+) Blackening of the agar slant (+) Group D and enterococci MANUAL: For gamma-hemolytic (-) NO blackening (-) S. pyogenes, S. agalactiae, S. pneumoniae, Viridans Group D = grows readily on BEA, hydrolyzes the esculin = DARK BROWN streptococci (denotes bile tolerance, ability to hydrolyze esculin, positive reaction_ Incubate: 35o C in ambient air for 48 hours Confirm identification with lancefield serological typing test OPTOCHIN (TAXO P) TEST (S) Zone of inhibition > 14 mm (S) S. pneumoniae MANUAL: with 6 mm disk For alpha-hemolytic (R) S. pyogenes, S. agalactiae, Viridans streptococci, Group D Enzyme: optochin (R) no zone of inhibition streptococci, Enterococcus (EQUIVOCAL)