Microsoft Presentation 2 Identification Of Clinically Significant Bacteria X1000 MARIONcompressed
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Identification of Clinically Significant Bacteria Marion Yuen Laboratory Diagnosis of Infectious Diseases: From Basics to Molecular Methods Workshop Friday 18 March 2011 Westmead Education & Conference Centre The Royal College of Pathologists of Australasia has received Australian Government funding under the Specialist Training Program Difficult Gram Positive Cocci Streptococcus mutans Alloiococcus otitidis Gemella haemolysans Helcococcus kunzii Tests that you need to perform Gram stain, haemolysis, motility, catalase Morphology in BHI Broth, 24hrs @ 37 °C ?clusters or chains Vancomycin susceptibility Bile esculin PYR, LAP, ADH, Aesculin, hippurate – available as rapid disc tests (Remel, Rosco) or part of commercial kit Growth in 6.5% NaCl, Tween 80 broths and @ 45°C Gas from MRS broth Streptococcus mutans Streptococcus mutans • Text Streptococcus mutans grown in BHI broth x400 phase contrast gs x1000 small GPR/coccobacilli Streptococcus mutans - HBA 24 & 48 hours Alloiococcus otitidis Key features GPC regular size, in pairs, tetrads, clusters Slow-growing (48-72hrs) due to lipid requirement Strict aerobe and asaccharolytic (unusual for GPC) Non-haemolytic to α-haemolytic with age Identified by API 20 Strep & ID 32 Strep Key tests: Catalase +/+w, PYR+, LAP+, 6.5% NaCl+ (slow), 45°C-, growth on BE but aesculin-, poor or no growth on CA Alloiococcus otitidis gs x1000 Always check morphology in BHI broth (I know I go on about this!!) Alloiococcus otitidis HBA @ 48hrs Gemella haemolysans Key features Gram-variable cocci in pairs, clusters & small chains Slow-growing CO2 enhances growth – G. morbillorum prefers to grow anaerobically, G. haemolysans prefers to grow aerobically Colonies are α-haemolytic or non-haemolytic Growth stimulated by Tween 80 May be confused with NVS but not B6 dependent Key tests: PYR+/V (requires heavy inoculum), LAP (V), BE-, 45°C-, Gemella haemolysans NO2+, Gemella morbillorum NO2- Gemella haemolysans – gs x100 cocci are often decolourised Helcococcus kunzii Key features Large irregular GPC in clusters – “Aerococcus-like” Non-haemolytic – some strains weakly α-haemolytic Lipophilic – growth stimulated by Tween 80 Not included on all commercial kits/system databases, but ID 32 Strep gives a profile 4100413 – “doubtful” A. viridans Key tests to differentiate from Aerococcus: LAP-, PYR+, aesculin+, hippurate-, NG @ 45°C, Tween 80 stimulation Note: Follow manufacturer’s instructions for rapid disc tests Helcococcus kunzii – gs x1000 cocci are large and in clusters Always check morphology in broth (BHI) – DO NOT RELY ON GRAM STAIN FOR MORPHOLOGY – see next slide! GPC’s in large clusters prepared from HBA plate gs x1000 - Same organism grown in BHI broth for 24 hrs Helcococcus kunzii HBA @ 24hrs Helcococcus kunzii HBA @ 48hrs Difficult Gram Positive Rods Anaerobes Clostridium tertium Features Slender long Gram-positive rods with oval, terminal spores anaerobically but not aerobically Aerotolerant Clostridia: C. tertium, C. histolyticum, C. Carnis Mis-identified as lactobacillus if spores not detected or Bacillus species if growth conditions not examined. Key tests: Bacillus spp: cat +, sporulates aerobically C. tertium: cat -, sporulates anaerobically Clostridium tertium – gs x1000, terminal, oval spores Clostridium tertium HBA, CO2 Clostridium tertium HBA, ANO2 Clostridium septicum Key features Gram-positive rods – medium to large, some “lemon” shaped rod forms, staining often uneven Spores – oval, central to subterminal, distends cell Strict anaerobe, saccharolytic Metronidazole = S Catalase negative Highly motile – swarms over plate in 24hrs! Must distinguish from C. sporogenes – lipase, lactose, mannose, enzyme profile on Remel RapID ANA II or other commercial kit (PRO & PYR enzymes) Clostridium septicum gs x1000 central to subterminal spores, lemon-shaped cells Swarming over HBA plate in 24 hrs Bifidobacterium longum Special features Habitat intestinal tract of man and animals Anaerobic GPR - some species are aerotolerant Curved rods, rudimentary branching and “bifid” forms, dog bone, long club forms - Gram stain morphology is the key! Generally resistant to MTZ Fermentative >30 species – B. dentium (previously B. eriksonii only pathogen) “Bifid” form Bifidobacterium spp. gs x1000 Bifidobacterium spp. some species are aerotolerant Check growth conditions! Dermabacter hominis Key Features GPR – small coccoid to tear drop shaped coryneform rods Colonies white to grey, shiny, can be sticky Fermentative metabolism Identifies on API Coryne Unusual reactions: LDC +, ODC +, aesculin + Cardiobacterium hominis - HBA @ 48hrs Dermabacter hominis gs x1000 Dermabacter hominis Dermabacter hominis 48hrs HBA Actinomyces species GPR, coryneform, curved, irregular, some branching or extensive branching Note: Some newer Actinomyces spp. show very little branching and may appear coryneform Colony appearance varies with species Non-haemolytic, α-haemolytic or β-haemolytic Hints that an isolate may be an Actinomyces are: -fermentation of xylose, lactose or aesculin hydrolysis -growth conditions API Coryne ID: Microbacterium/Cellulomonas, G. vaginalis Actinomyces turicensis gs x1000 Actinomyces turicensis Close-up Actinomyces turicensis 24 & 48hr HBA Actinomyces israelii – gs x1000 – microcolony grown in BHI broth Actinomyces neuii spp. neuii gs x1000 Note – irregular GPR, curved rods with tapered ends Actinomyces spp. vary in their Gram stain appearance Actinomyces odontolyticus Note – anvil shaped rod & small rudimentary branch node Corynebacterium sundsvallense Special features GPR - irregular pleomorphic rods Catalase + Fermentative Non-haemolytic Non-pigmented Non-motile No substrate or aerial mycelium Colonies resemble aerobic actinomycete but growth conditions are consistent with Corynebacterium C. Sundsvallense gs x1000 showing swellings at end of rods C. Matruchotii gs x1000 - whip handles Corynebacterium sundsvallense – HBA, 72hrs C. durum, C. matruchotii & Rothia dentocariosa share similar characteristics Arcanobacterium haemolyticum Key features Gram-positive coryneform & irregular, curved rods Colonies 0.5mm smooth, dry, whitish colonies at 24hrs β-haemolytic colonies best observed @ 48hrs 3 medically relevant species: A. haemolyticum, A. pyogenes, A. bernardiae Identifies well on API Coryne key tests to differentiate species - gelatin, xylose, glycogen A. haemolyticum – gs x1000 irregular gram positive rods Arcanobacterium haemolyticum - showing β-haemolysis at 48hrs Microbacterium oxydans Key features GPR regular or pleomorphic, curved Colonies moist white-cream or yellow with age Motile or non-motile Oxidative metabolism but genus is a mixture of both oxidative & fermentative species – makes ID confusing API Coryne usually gives a clue (mannitol +, Aesc +) but check growth conditions Microbacterium spp. - gs x1000 Microbacterium spp. HBA @ 48hrs pigment varies from white, grey to yellow Aerobic Actinomycetes Before we get started Questions you need to answer Is the isolate a strict aerobe (oxidative) or facultative anaerobe (fermentative) Is the organism a GP branching rod or irregular nonbranching rods Do the rods stain poorly Is there a substrate mycelium and aerial hyphae Is there substrate mycelium only Is there neither substrate nor aerial hyphae Is the isolate acid fast by ZN or modified ZN 41 First - Why are some bugs acid fast? Cells that are acid fast contain mycolic acids (large group of long chain fatty acids of varying length) The amount of mycolic acids will confer varying degrees of resistance to chemicals, permeability & acid fastness (stain binds to cell wall mycolic acids) Mycolic acid chain length (carbon number) 0 20 30 40 50 60 70 80 90 Dietzia Nocardia Tsukamurella Mycobacterium Coryne Rhodococcus Gordonia Secondly – Aerial hyphae & substrate mycelium Are you sure you know what you’re looking for and how to go about it? Looking for Substrate and Aerial Hyphae? Place culture plate on stage - must use clear medium e.g. NA, MHA, SAB Drop condenser to increase contrast Start with x10 objective to locate individual colonies Change to x20 objective to examine structures more closely, BUT don’t end up in the agar! Rhodococcus equi Special features GP rods – coryneform, jointed or rudimentary branching – morphology varies according to progression of the rod-coccus cycle Non-motile Oxidative metabolism Colonies – translucent mucoid, salmon pink with age Marked rod-coccus cycle but no substrate or aerial hyphae May be partially acid fast (Modified ZN) Identifies on API Coryne Note: mucoid pink colonies could be Roseomonas spp. or other pink oxidative GNR – check gram & do vancomycin or string test Rhodococcus equi rod-coccus cycle - note variation in morphology Jointed rods Rhococcus equi – showing oxidative growth pattern No substrate or aerial hyphae Compare this image to the next slide Nocardia spp. – x400 Early colony development showing substrate mycelium Dark aerial hyphae Light substrate mycelium Nocardia spp. NA x10 - note substrate & aerial hyphae Important Note Don’t be tempted to look for substrate mycelium or aerial hyphae too soon! Check out the next slide ……. Corynebacterium sundsvallense – MHA, 4 days, x100 Nocardia species Key features GP beaded branching rods, fragment to non-motile rods coccobacilli Colonies adherent, dry, chalky, heaped & folded with age Strict aerobe Acid-fast - modified ZN stain (Kinyoun) Substrate mycelium & aerial hyphae Key tests: Lysozyme = R, speciate by antibiotic susceptibility, assimilation reactions, amino acid hydrolysis reactions & Arylsulfatase test - (+ for N. nova only) Complex “birch tree” branching Nocardia spp. - NA x100 Nocardia spp. branching GPR branches are often at right angles Nocardia spp. gs x1000 Fragmentation to rods & coccoid elements Nocardia spp. – Modified ZN stain showing partial acid fast coccoid-rod elements Nocardia spp. NA slope @ 7 days Pigment varies from chalk white, salmon pink, orange Colonies are adherent, dry, heaped and folded with earthy odour Streptomyces species Key features Long filamentous gram positive filaments with minimal branching Colonies khaki grey, heaped, folded, adherent, become chalky white with age, earthy odour Strict aerobe NOT acid-fast Substrate mycelium, occ. aerial hyphae that form chains of conidia No Fragmentation occur Lysozyme = S, biochemically difficult to speciate – perform16SrRNA gene sequencing 57 Streptomyces griseus, x100 - MHA at 48-72 hours Streptomyces spp. gs x1000 Long filamentous hyphae with less branching than Nocardia spp. Streptomyces spp. 24 & 48hr, HBA Colonies become dry & chalky white as aerial hyphae form Oerskovia species Key features Irregular GPR, branching filaments Colonies - smooth, glistening, bright yellow Facultative anaerobe, Fermentative NOT acid fast Substrate mycelium that fragments into motile elements but NO aerial hyphae. This differentiates L. aquatica and Microbacterium from Oerskovia API Coryne identifies Oerskovia Key tests: hydrolysis of casein, gelatin, xanthine, hypoxanthine Substrate mycelium, no aerial hyphae Oerskovia spp. - NA, x1000, Substrate mycelium only Oerskovia spp. Facultative anaerobe Excludes Leifsonia aquatica Bright yellow pigment 2 species – Oerskovia turbata Cellulosimicrobium xanthinolytica Tsukamurella species Key features Irregular GPR – no obvious branching, may stain gram variable Rough & highly wrinkled colonies in 1-2 days Pigment varies with species – wh, cr, yell, orange Aerobic Partially & weakly acid fast by ZN & Modified ZN No substrate or aerial hyphae Lysozyme = R, 3 Day Arylsulfatase test –, Urea + Tsukamurella spp. – gs x1000 Tsukamurella inchonensis - HBA @ 72hrs Oxidative metabolism – no growth ANO2 Tsukamurella spp. Whole colonies - NA plate at 4 days “spider-like” colonies Tsukamurella colonies, NA plate at 7 days – closer view This is not substrate or aerial hyphae! – only sticky rods Mycobacterium fortuitum Mycobacterium abscessus Key features Faint staining, gram-variable filaments & curved rods, “ghost” cells (branching filaments can occur with M. fortuitum) Slow-growing (3-5 days) aerobe Colonies non-pigmented, buff to yellow, smooth or dry Acid-fast but may be weak or partial Members of M. chelonae complex – highly resistant to antimicrobial therapy Close up – colonies can be dry or moist Simple “fir tree” type mycelium Mycobacterium fortuitum – 3 months on NA Let’s have a break and take in the view! Cardiobacterium hominis - HBA @ 48hrs Gram Negative Rods Bordetella holmesii Key features Small to medium slender GNR, some curved rods Non-haemolytic, slow growing aerobe Oxidase -, Catalase V, MAC + but growth is slow Oxidative Non-motile Non-reactive - browning on tyrosine agar Not on database of commercial ID kits/systems 74 Bordetella holmesii gs x1000 small-medium slender GNR some curved mis-identifies as Acinetobacter spp. BUT Gram stain morphology is not plump GNCB! Browning on Tyrosine agar Bordetella holmesii growth O2 & CO2 but not anaerobic typical of oxidative organisms Moraxella atlantae Key features Plump GNCB, often stains gram variable Strict aerobe - Oxidative Growth stimulated by bile salts (MAC growth is better or equal to HBA) Non-motile buy twitching has been observed (pili) OX+ (excludes Acinetobacter spp.), CAT+, asaccharolytic M. osloensis – some strains tributyrin +, ß-Lactamase +, vancomycin, R Moraxella atlantae gs x1000 Plump GNCB Cells can stain Gram variable & are very coccoid – always check with penicillin challenge & String Test Important feature is the colony appearance Colonies are clear & tiny @ 24hrs, pitting and non-pitting forms do occur Moraxella atlantae @ 4 days showing spreading flat growth 79 Roseomonas gilardii Key features Plump coccoid to oval rods in pairs or short chains Resists decolourising Colonies mucoid and pale rose pink Most species grow on a broad range of media & at 30-42°C Oxidase +w/-, Urease + Roseomonas spp. are not on database of API 20 NE - identify as Methylobacterium spp. Confused with N. gonorrhoeae - grows on TM medium 80 Roseomonas spp. – gs x1000 Roseomonas cervicalis HBA, 48hours NA plate showing mucoid colonies with pale rose pigment 82 SAB & HBA, 30°C at 5days Methylobacterium spp. HBA, 37°C at 5 days Methylobacterium spp. - gs x1000 Terrible gram – but this is what it can look like! 84 Capnocytophaga ochracea Key features Slender, fusiform rod, tapered ends Capnophilic Gliding motility Yellow-orange “ear wax” pigment (swab technique)and pink-lilac sheen Colonies fringed to spreading, beaten copper look (C. ochracea) Bluish-grey, entire colonies (C. canimorsus) Key distinguishing tests: OX, CAT, ADH Capnocytophaga ochracea gs x 1000 Spreading growth, beaten surface of colonies Capnocytophaga ochracea HBA @ 48hours Streptobacillus moniliformis Key features One of two agents of Rat-bite fever (also Spirillum minus – Sodoku) Recovered occasionally from blood culture with no added SPS Check patient history - ?rat bite, pet rat, drug abuse Unusual Gram stain – ‘string of pearls’, chains, filaments, swellings Non-haemolytic to weak alpha with age Non-motile Fermentative metabolism Identification – use a rapid substrate method e.g. ID 32 STREP or similar Streptobacillus moniliformis gs x1000 filamentous GNR with swellings Vibrio vulnificus Special features Cause of wound infections & primary septicaemia following ingestion of oysters Curved & straight GNR OX+/Fermenter Growth on TCBS = green Key tests: O/129 = S Salt tolerance to 6% (65%) Fermentation of sucrose -, salicin +, cellobiose +, lactose + >75% 90 Vibrio vulnificus Green colonies on TCBS at 24 hrs Vibrio vulnificus colonies on HBA at 24 hrs Vibrio vulnificus – Salt Tolerance 92 Vibrio parahaemolyticus Special features Cause of acute GE – consumption of contaminated seafood worldwide Curved GNR OX+/Fermenter TCBS = green Key tests: O/129 = S/R, salt tolerance to 8% (most strains), fermentation of sucrose -, salicin -, lactose -, cellobiose -, urea + (50% of strains) The “HACEK” Group What defines this group of organisms? Gram stain morphology Cultural characteristics – e.g. β-haemolysis i.e. K. kingae, pitting colonies, no growth on MAC, colony appearance i.e. dry, adherent, mixed appearance, slow growth rate Initial requirement for CO2 and X factor (lost on subculture) Key biochemical characteristics – e.g. indole (C. hominis) Best identified using a rapid substrate method Cardiobacterium hominis Key features Faintly stained Gram-negative regular rods “Comets” and rosette arrangements Colonies whitish, shiny, may pit the agar Key test: Fermentative, oxidase + cat -, indole + Differentiate from indole + Suttonella (Kingella) indologenes 95 Cardiobacterium hominis gs x1000 note ‘comet tails’ Eikenella corrodens Key features Faintly stained slender, straight sided, very regular gram-negative rods May be X-dependent on isolation (ignore this) OX +, CAT Pitting and non-pitting colony forms Key tests: Asaccharolytic, NO3 +, ODC +, LDC + Cardiobacterium hominis - HBA @ 48hrs 97 Eikenella corrodens - gs x1000 98 Eikenella corrodens - HBA CO2 @ 48hrs Kingella kingae Key features Plump Gram-negative coccobacilli in pairs & short chains, parallel rows & “railway tracks” “Soft” β-haemolysis on HBA Acid from glucose & maltose – has been confuse with N. meningitidis Associated with bone andhominis joint disease Cardiobacterium - HBAin@children 48hrs 100 Magnified view Kingella kingae Cardiobacterium hominis - HBA @ 48hrs gs x1000 Cardiobacterium hominis - HBA @ 48hrs Kingella kingae, HBA, CO2 at 48hrs Aggregatibacter aphrophilus Key features Small Gram negative coccobacilli Capnophilic Strong α-haemolysis on HBA Culture may look mixed Initial requirement for X factor Distinguish A. actinomycetemcomitans, catalase +, from A. aphrophilus, catalase - Aggregatibacter aphrophilus gs x1000 A. aphrophilus now includes A. paraphrophilus Cardiobacterium hominis - HBA @ 48hrs (V dependent strain) 104 Aggregatibacter aphrophilus, HBA@ at 48hrs 48hrs Cardiobacterium hominis - HBA Aggregatibacter actinomycetemcomitans Key features Tiny Gram-negative coccobacilli Capnophilic Colonies adherent, white, dry Fermentative Distinguish from Brucella spp. – similar Gram stain Distinguish from A. aphrophilus (Catalase Cardiobacterium hominis - HBA @-)48hrs 106 Aggregatibacter actinomycetemcomitans gs x1000 Brucella melitensis gs x1000 Cardiobacterium hominis - HBA @ 48hrs Aggregatibacter actinomycetemcomitans – HBA plates 48hours. Cardiobacterium hominis - HBA @@48hrs Growth conditions for B. melitensis would be reversed – it is a strict aerobe! Anaerobiospirillum succiniciproducens Key features Important to recognise that this is not a campylobacter! Gram-negative helical rods with rounded ends Motile +++ - corkscrew Strict anaerobe – not microaerophilic Colonies clear, flat, spreading Catalase -, Oxidase – Key tests: Glucose, indoxyl acetate, nitrate, urea, H2S Cardiobacterium hominis - HBA @ 48hrs in SIM media Anaerobiospirillum succiniciproducens gs x1000 Swarming on HBA plate after 48 hrs Cardiobacterium hominis - HBA @ 48hrs anaerobic incubation A final cautionary note~ Increasingly sophisticated identification methods are becoming available, improving accuracy and turn around times. We welcome these advancements which result in improved patient outcomes. However we must not neglect basic microbiology skills in the belief that automation can replace them – we must recognise a microorganism of significance before it can be identified – some detective work and sound microbiology are required. Cardiobacterium hominis - HBA @ 48hrs Thank you for listening Any Questions?
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