The Phenotypic Identification of Anaerobic Bacteria

Diane© by M. author Citron R.M. Alden Research Lab ESCMID OnlineCulver LectureCity CA Library Appropriate specimens

• Blood – (RX with ineffective ABX = higher mortality) • Other normally sterile sites • Dental/sinus – aspirate or BX • Lung – aspirate or BX, thoracentesis • Abdomen – paracentesis, asp of abscess, surgical specimen© by author • FG tract – laparoscopy, surgical, aspirate • Bone – aspirate, BX (not sinus tract) ESCMID• Soft tissue Online – surgical, Lecture BX, asp, Library drge

Transport of Specimens

• Aspirates – anaerobic transport tube • Tissue specimens – anaerobic bio bag, anaerobic transport tube with medium • Swab specimens (not first choice) should be placed immediately into anaerobic transport medium – should be cultured within 24h for optimal recovery • eSwabs are excellent for maintaining viability

 Boiled free of molecular oxygen

 Reducing agent then added

 Autoclaved anaerobically

 Dispensed© anaerobically by author

 Stored anaerobically ESCMID Online Lecture Library  Not the same as “reduced” plates Identification of anaerobes

• Group level identification – Selective and differential media – Colony morphology – Gram stain cell morphology – Special potency antibiotic disks – Nitrate disks © by author – Rapid tests- catalase, spot indole, urease ESCMID Online Lecture Library

Selective and differential media

• Laked blood with kanamycin and vancomycin Brucella agar (LKV) • Inhibits gram-positives and enterics • Grows Bacteroides, Prevotella, Alistipes, some fusobacteria (and Clost. innocuum!)© by author • ESCMIDEnhances pigmentationOnline Lecture of Library Prev. melaninogenica group • Porphyromonas do not grow Selective and differential media

• Bacteroides bile-esculin- gentamicin agar (BBE) • Rapid presumptive ID of B. fragilis group, Alistipes and Bilophila. © by author • Some strains of Fusobacterium mortiferum and F. varium may grow.ESCMID (bile resistant) Online Lecture Library • Enterococci highly resistant to gentamicin will grow Chromogenic Bacteroides compared to bile-esculin agars

Tierney D et al Anaerobe 2016

Anaerobe plates

P. intermedia

B. fragilis Spot indole test p-DMACA reagent

Colonies can also be rubbed onto filter paper moistened with reagent Blue color is + © by author ESCMID Online Lecture Library Rapid urease test (disks, tablets)

preformed enzyme; incubate aerobically, read 1-2h

Bile Kana Van Col Cat Nit B. frag grp R R R R + - Prevotella S R R V - - Porphyromonas S R S R - - Fusobacteria V S R S - - Bilophila © R by author S R S + + Desulfovibrio V S R R -+ + C.ureolyticusESCMID grp. Online V Lecture S R Library S -+ + Other bile-resistant GNB

• Alistipes putredinis (formerly Bacteroides), A. onderdonkii, A. shahii. – Indole-positive, catalase-neg – Growth on BBE – gray colonies, esculin neg • Odoribacter splanchnicus (Bacteroides) – Indole-positive,© catalase- by authorneg – Growth on BBE – weak or neg • Both cultured from intra-abdominal ESCMIDinfections, occasionally Online Lecture other sites Library Non-pigmented Prevotella species

IND ESC b-NAG a-FUC b-XYL Cat P. buccae - + - - + - P. oris - + + + + - P. buccalis - + + + - - P. enoeca - V + + - - P. oralis - + + + - - P. oulorum - © by + author + - - - P. bivia - - + + - - P. disiens ------P. heparinolyticaESCMID Online + + Lecture + + Library + - Pigmented Prevotella species

ind lip a-gal b-gal NAG P. corporis - - - - - P. denticola - - + + + P. loescheii - - + + + P. melaninogenica - - + + + P. intermedia + + - - - P. nigrescens © + by author +/- - - - P. pallens + - - - - P. tanneraeESCMID Online - Lecture - - Library + + Pigment, fluorescense, lipase

species bile NO3 ind lipase esc cells nucleatum S - + - - s,pt necrophorum SR - + + - r,pleo naviforme S - + - - boat gonidiaform S - + - - r,gonidia russii S - © by- author - - r canifelinum S - + - - pt mortiferumESCMID R Online - - Lecture - +Library pleo varium R - +- -+ - r,pleo Fusobacterium nucleatum

• S to kana, colistin disks; R to vanco • indole positive; lipase negative • slender rods with pointed ends • several different colony types; subspecies • Isolated from all types of infections and all areas of the© body by author • polymicrobial infections or single isolate ESCMID• Associated Online with colorectalLecture cancer, Library preterm delivery F. nucleatum

• S to kana and colistin disks ; R to vanco • indole and lipase positive; beta-hemolytic • cells have rounded ends or may be very pleomorphic • important pathogen in Lemmiere’s disease and postanginal sepsis in young people (deep vein thrombosis,© metastatic by author thrombi) • Cause of persistent sore throat with systemic ESCMIDsymptoms (PII- Online10 Lecture Library Fusobacterium necrophorum

•

• S to kana, colistin disks • bile-resistant • F. mort is indole neg; F. varium esculin, ONPG pos • F. varium is indole posneg; esculin, ONPG neg • pleomorphic cells • occasionally isolated© by author

from intraabdominal and otherF. mortiferum lowerESCMID body infections Online Lecture Library

Campylobacter ureolyticus-like group • Includes C. ureolyticus, C. gracilis Sutterella, some Campylobacter spp., • GNB sensitive to kana, colistin, R to vanco • Small transparent/translucent colonies • Pitting of agar© is by variable author • Most reduce NO3 ESCMID Online Lecture Library Campylobacter ureolyticus-like grp Kana-S, Vanco-R, Col~S

URE MOT CAT BILE CO2 Camp. ureolyticus + - -+ - - Camp. gracilis - - - - - Camp. rectus - + - - - Bilo. wadsworthia + - + R - Sut. wadsworthen. - - - R - Dial. pneumo. Col-R - © by - author - - - Desulfovibrio Col-R -+ +/- -+ -R - Eiken. corrod. - - - - + ESCMID Online Lecture Library ( Use disk test for bile resistance) Campylobacter ureolyticus

Transparent colonies, pitting of agar

• Original isolates from appendicitis (1990) • Later from many other sources (chronic sinusitis, cholesteatoma, pleural fluid, liver abscess,© blood) by author • Grows on BBE, black centersESCMID in colonies Online (H2S +)Lecture Library • Catalase very positive !!!!! • B-lactamase pos, resistant to many b-lactams

Desulfoviridin test and H2S

• Desulfoviridin rapid test: – Cell paste on cotton swab – Add drop of 2 N NaOH – Look for red fluorescence under UV light – Bilophila also positive • H2S pos in SIMS© mediumby author ESCMID Online Lecture Library Desulfovibrio species

Mo cat ind nitr Bile ure desul D. species: tili disk a foviri ty din piger - - - - R - + fairfieldensis + + - + R - + © by author desulfuricans + - - + S + + ESCMID Online Lecture Library vulgaris + - + - R - +

Warren el al J Clin Microbiol 2005 43:4041 Gram-negative cocci

Genus NO3 catalase glucose

Veillonella + V - Acidaminococcus - - - Megasphaera - - + © by author (Dialister resembles Veillonella – tiny GNB, ESCMIDNitrate-neg, Online Col - R)Lecture Library

Gram-positive cocci

SPS Cat Ind Ure a-glu P. anaerobius S - - - + P. asaccharolyticus R -+ + - - P. harei R -+ + - - P. micra © by R(s) author - - - - F. magna R -+ - - - A. prevotiiESCMID Online R Lecture - - Library+/- + A. tetradius R - - + + MANY NEW SPECIES HAVE BEEN DESCRIBED

GPAC

• Parvimonas micra

Peptoniphilus asaccharolyticus

• S. intermedius, S. constellatus, G. morbillorum previously classified as Peptococcus and Peptostreptococcus sp. • Abiotrophia and Granulicatella initially may appear to be anaerobes but

eventually will grow in CO2 • Frequently recovered© by fromauthor abscesses • Resistant to metronidazole, usually ESCMIDsusceptible toOnline penicillin Lecture Library Gram-positive bacilli

• Check for spores to rule out clostridia • Some clostridia stain gram-negative (will be vanco sensitive) • Some clostridia have rare or no spores • Colony morphology important - frequently colonies will have© spreadingby author or irregular edges ESCMID Online Lecture Library Ethanol spore test

• Suspend colonies from older (2-4 days) plate culture into any broth • Mix with equal volume of ethanol • Subculture to BA after 10-30 minutes • Growth indicates presence of spores • Subculture untreated© by brothauthor as growth control

ESCMID Online Lecture Library Most frequent clinical isolates of Clostridium spp. (IUMC)

• C. perfringens 20% • C. clostridioforme grp 16% • C. innocuum 15% • C. ramosum 14% • C. difficile © by author11% • <4% C. butyricum, C. cadaveris, <1% C. sporogenes, C. bifermentans, C. glycolicum, C. tertium, C. septicum,ESCMID C. sordellii, Online C. paraputrificum,Lecture Library C. symbiosum, C. subterminale, C. barati MCM 8th Ed Characteristics of C. perfringens

• Box car shaped GPB • Double zone of beta-hemolysis on BA • Lecithinase positive (alpha toxin) © by author • ProducesESCMID abundant Online gas Lecture Library in liquid media (blood culture bottles?) Clostridium innocuum

• Resistant to cefoxitin, other cephs, vancomycin (MIC= 8-16), some strains to clindamycin, quinolones • Grows on CCFA medium – resembles C. difficile • Disks- resistant to Ka, Va, Cl • Misidentified in preformed enzyme kits ( often as C. subterminale)© by author • Gram variable rod, occ large ESCMID terminal spores Online Lecture Library • Isolated from IA infections, blood Clostridium ramosum • Gram-variable long slender rod • Chains in broth media • Spores are rarely visible – terminal • May be resistant to antimicrobials • Identify using enzyme kits, biochemicals© by author ESCMID Online Lecture Library Clostridium clostridioforme grp

• Typically stains Gram-negative • Spores are rarely seen • Sensitive to vanco and kana disks • One of most frequently encountered clostridia • ~30% produce beta-lactamase;© by author most generally more resistant to antimicrobials • IdentifyESCMID with enzymeOnline kits Lecture or biochems Library • New species: boltae, H. hathewayii, citroniae, aldenense Clostridium septicum

• Swarmer; subterminal spores; indole-neg • Bacteremia associated with cancer of large bowel malignancies • Portal of entry – ileocecum • Myonecrosis, gas gangrene • Underlying conditions -leukemia, lymphoma, diabetes, © by author • Susceptible to usual antimicrobials •ESCMIDToxins not eliminatedOnline Lecture with abx therapyLibrary • High mortality

Clostridium septicum (swarmer)

PEA inhibits swarming Gram stain of tissue

catalase nitrate indole urease Propionibacteria + +- +- - Actinomyces -+ +- - -+ Eggerthella lenta +(w) + - - “Eubact. grp.” © - +by author - -+ - Lactobacilli - - - - BifidobacteriaESCMID Online - Lecture - Library - - Propionibacterium acnes

• Frequent skin contaminant in blood, csf cultures • Occasionally pathogenic (shunts, implants, post- op cultures from eye) • Relatively slow-growing – broth culture up to 14d • ID based on pos rxns for catalase, indole, ©nitrate by author • Acne strains may be R to tetraESCMID and macrolides Online Lecture Library • All strains R metronidazole API 20A

• Identification of asaccharolytic organisms • Growth in the system not required • Rapid (15 ©min- by 4 author hr) • Less expensive than conventional ESCMID Online Lecture Library RapID ANA II

Be careful w/ arabinosidase (3rd © by author test) B frag = neg ESCMIDB. caccae=pos Online Lecture Library Do not overread yellow Rapid ID 32A

© by author ESCMID Online Lecture Library Disadvantages of Preformed Enzyme Kits • “One size fits all” – 50-120 very different organisms in data base – all tests not appropriate for every species (coagulase on E. coli??) – The more tests in the kit, the greater the opportunity© by author to generate a non- matching profile number ESCMID– Most Onlineproblems Lecturewith indole-pos Library Bacteroides, GPB and clostridia