Actinomyces – Problem Or Not?

Actinomyces Systematics, laboratory diagnosis and clinical aspects

Trefor Morris, UK Anaerobe Reference Unit, Cardiff UK Anaerobe Reference Unit

• Serves the whole of the UK • One of a select few world-wide • Remit of the ARU includes: 1. Identification & susceptibility testing 2. C. difficile ribotyping and surveillance 3. Clinical & technical advice 4. Maintenance of a reference collection of clinical isolates© c.30,000by author 5. Training ESCMID6. R&D, product Online evaluations Lecture Library

Actinomyces

• Non-sporing gram positive bacilli • Facultative anaerobes • Metronidazole resistant • Not acid-fast • Fermentative • Catalase variable© by author • Indole negative ESCMID• ~40 species, Online ~20 affecting Lecture humans Library Levels of Bacterial Classification • Kingdom – All bacteria are in the Prokaryotes • Subkingdom – Bacteria, Archea • Phylum – e.g. Firmicutes, Actinobacteria • Class – e.g. ‘Clostridia’, ‘Actinobacteria’ • Order – e.g. Clostridiales, Actinomycetales • Family – e.g. Actinomycetacae • Genus – This is the level that most bacteriologists deal with • Species - © by author • Sub-species - ESCMID Online Lecture Library Classification of Actinomyces

• Phylum = Actinobacteria • Class = Actinobacteria • Order = Actinomycetales • Family = Actinomycetaceae • Genus = Actinomyces © by author ESCMID Online Lecture Library 1990 2001 human and animal species • A. hyovaginalis • A. bowdenii • A. bovis • A. canis • A. israelii • A. catuli • A. meyeri • A. europaeus • A. naeslundii • A. funkei • A. odontolyticus • A. graevenitzii • A. viscosus • A. marimammalium • A. denticolens • A. neuii (2 subspecies) • A. georgiae • A. radicidentis • A. gerencseriae • A. radingae • A. hordeovulneris © by author• A. suimastitidis • A. howellii • A. turicensis • A. slackii • A. urogenitalis ESCMID Online Lecture Library Additional species recognised by 2007

From human sources Animal sources A. cardiffensis A. canis A. dentalis A. catuli A. funkei A. coleocanis A. hongkongensis A. marimammalium A. nasicola A. ruminicola A. oricola A. suimastitidis A. radicidentis A. vaccimaxillae A. urogenitalis © by author

ESCMID 2012 - Actinomyces Online Lecturenaturae - environmentalLibrary

Not to be confused with …

• Aerobic 'Actinomycetes' – Nocardia – Streptomyces • Mycobacteria • Corynebacteria • Other facultative anaerobic NSGPB (Propionibacteria, Lactobacilli, Bifidobacteria, Gardnerella…) © by author ESCMID Online Lecture Library …but closely related to…

• Arcanobacterium • Actinobaculum • Varibaculum • Mobiluncus © by author ESCMID Online Lecture Library Normal habitats (Man and mammals)

• Oral cavity (tongue, plaque, periodontal)

• Gastro-intestinal tract (small numbers)

• Genito-urinary tract • ? Lipid rich areas© byof skin author ESCMID Online Lecture Library Actinomycosis - historical

Actinomycosis in humans - Wolf & Israel, 1891

‘LumpyESCMID jaw’ in Onlinecattle - Bollinger Lecture / Harz Library 1879 Spectrum of Disease Classically: cervico-facial Less commonly: thoracic, abdominal, pelvic sites Rarely: CNS, long bones, joints

Some very rare presentations:

Paediatric osteomyelitis Robinson et al, 2005. Pediatr Infect Dis J. 24:365-9. Midfacial osteomyelitis in a chronic cocaine abuser Talbott et al, 2001. ©Ear byNose authorthroat J. 80:738- 43. Gastric actinomycosis after gastric bypass ESCMIDFernandez-Acenero Online et al, 2004. Lecture Obes Surg. Library 14:1012-5 . Actinomycosis of the head and neck

Swelling Osteoradionecrosis Chronic submandibular (ORN) of jaw abscesses with draining sinus tracts Other© sites by include: author Permanent disfigurement Brain abscess May be fatal if not treated Canaliculitis ESCMID OnlineDental abscess Lecture Library

Actinomycosis – other sites Thoracic – lung, pleura, chest wall, spine

Abdominal – stomach, liver, appendix, gallbladder, abdo. wall

Pelvic – tubo-ovarian, intra-uterine, bladder, colon, psoas

Other – 'punch' actinomycosis (fist), skin, long bones

• Association of actinomyces-like organisms (ALOs) in Cx smears of IUCD-users noticed by cytologists in 1970s

• Association has been proven but ? colonisation

• Significance re. pelvic disease is much debated

• ? Removal of IUCD - infection risk must© be by balanced author against risks of unwanted pregnancy, other contraceptives, re- ESCMIDinsertion of IUCDOnline Lecture Library IUCDs • ~20% colonisation with Actinomyces • Usage = common, pelvic infection = rare • ? Significance of Actinomyces spp. • BUT ? best specimen short of surgery

British Faculty of Family Planning and Reproductive Healthcare recommendations, Cayley et al (1998)

• Actino + symptoms = ©remove by IUCD,author treat, review • Actino – symptoms = counsel re. risks / symptoms, follow-up, ? leaveESCMID / remove / replaceOnline IUCD Lecture Library Agents of actinomycosis

Producing granules / filamentous microcolonies:

A. israelii A. gerencseriae (A. israelii serotype II) A. graevenitzii Propionibacterium propionicum (formerly Arachnia propionica)

Usually smooth colonies, diphtheroid cells:

©A. meyeriby author A. naeslundii / viscosus complex A. odontolyticus ESCMID Online? other Lecture species Library Actinomyces in other infections • Periodontal disease / dental A. naeslundii, A. odontolyticus, caries A. israelii, A. gerencseriae

• Superficial soft tissue A. turicensis, A. radingae, abscesses A. europaeus, A. funkei, A. • perianum, axilla, breast neuii, • pilonidal sinus Varibaculum cambriense • balanitis

A. naeslundii, A. odontolyticus, • Bacteraemia / endocarditis A. turicensis, A. funkei

• Chronic / complicated UTI © by authorActinobaculum spp., A. neuii • 'sterile' pyuria

Hall ESCMIDV (2008). Actinomyces – gatheringOnline evidence of Lecturehuman colonization and Library infection. Anaerobe 14:1-7. Non-classical Actinomyces species

Actinomyces turicensis • ? Flora of genital and GI tracts, skin • Common in superficial soft tissue abscesses: pilonidal, perianal, axilla, penile etc. • Rare cause of endocarditis

ESCMIDA. naeslundii Online LectureA. odontolyticus Library A. funkei A. neuii subsp. neuii subsp. anitratus • Habitats, clinical sources • ? Flora of skin similar to A. turicensis • From breast abscess etc.

• ? Oral flora • ? Habitat • ? Rare cause of actinomycosis, • Sources as 'classic' Actinos. intra-oral, thoracic • ? significance

Red under UV light

Dense filamentous microcolonies (eosinophilic) Surrounded by basophilic material (bacterial and host) Granulomas / cavitations / sinuses develop Bacteria protected from further host responses Protected from antimicrobial activity Similar to TB interaction© by author ESCMID Online Lecture Library Early actinomycosis

• Boil-like appearance Usually after trauma e.g. dental surgery, fist-fight Intra-oral effects

• Early colonisers of oral cavity • Adhere to teeth and bacteria – help form plaque • ?? Significant in© dental by authorcaries • Periodontal diseases •ESCMID Root canal infectionsOnline Lecture Library Actinomycosis – clinical summary

• Advanced cases are rare in the UK • Chronic, progressing slowly, spreading across fascia, disfiguring • Difficult to diagnose – may be mistaken for malignancy or TB • Prolonged / radical therapy required • 60% cervico-facial, 20% thoracic, 20% abdominal / pelvic© by author • relapse / treatment failure = common • ESCMIDMay be eventually Online fatal Lecture if not treatedLibrary Patient management • Surgery if necessary: drainage of exudate / debridement / excision of infected tissue • Long-term high-dose penicillin But…  Advanced disease may be difficult to treat  Relapses may occur months / years later  Vague symptoms,© slowby authorprogression • difficult to diagnose  Mis-diagnosis as cancer or TB is common ESCMID• may result Online in unnecessary Lecture therapy Library / surgery Laboratory diagnosis

• Appropriate specimen • Direct microscopy • Culture • Identification • Antimicrobial© by susceptibilities author ESCMID Online Lecture Library Problems with Specimens • If disease is not overt it may be difficult to get specimen

(CT guided needle biopsy, FNA may help)

• Diagnosis = Ca. Tissue in formalin = histology only

• Pus drained, but only swab sent to lab.

• Dry swab = worst case

• Unsuitable: oral swab,© by sputum, author vaginal swab (normal flora)

• LackESCMID of clinical Online info. = may Lecture not culture Library for Actinos Primary lab. problems

• Only found if looked for!

– May need prolonged incubation (5-10 days) – May only grow anaerobically – May not grow on selective (Neo) plates

• Invariably mixed • Plate microscope • Need selective media or hand lens can help – Metronidazole/Nalidixic acid – ? Mz / mupiricin – NOT neomycin or gentamicin – can use discs • Other orgs. may be inhibitory

• >10 days AnO2 incubation© by author • Unfamiliar with morphologyESCMID Online Lecture Library Good specimens - 1

Cerebral abscess pus

Direct Gram's stain of crushed granules

CultureESCMID = No growth Online @ 10 days. Lecture Prior antibiotics, Library old specimen . Good specimens - 2 Aspirate - liver abscess

Direct Gram's stains granule free pus © by author ESCMID Online Lecture Library Direct microscopy Culture (liver abscess pus) After 5-10 days anaerobic incubation

Non-selective blood agar, ideally FAA

Actino selective – 10mg/L MTZ, 30mg/L NAL

NOT Neo, Gent or Kana agars (Actino are sensitive)

o Incubate AnO2 @ 37 C for up to TEN DAYS

Infections usually polymicrobial© by author and other organisms grow faster  broth enrichment is rarely useful ESCMID Online Lecture Library Beware Bacterial inhibition!

Various Actinomyces spp. inhibited by:

•Enterococcus faecalis •Strep. pyogenes •Bifidobacterium spp. •Aerobic spore-bearer •Lactobacillus © by authorrhamnosus •Other Strep. spp. ESCMID Online Lecture•Prevotella Library bivia

Problems - Identification

• Must obtain pure culture • Slow-growing / gritty colonies • New species – not in textbooks / databases • Lack of familiarity / experience • ? Kits vs. alternatives – Sarkonen et al (2001) J Clin Microbiol – Santala et al© (2004) by author J Clin Microbiol – Kerttula et al (2005) Anaerobe ESCMID Online Lecture Library Evaluation of four commercial test systems for identification of Actinomyces and some closely related species

Santala, Sarkonen, Hall, Carlson, Jousimies-Somer, Kononen J Clin Microbiol 42:418-420

‘classic’ spp. ‘new’ species To species To genus RapID ANA II 40% 13% API RapID 32© A by 58%author 42% RapID CB Plus 26% 23% ESCMIDBBL CrystalOnline 65%Lecture Library49% ARDRA (Amplified rDNA Restriction Analysis)

Hall, O'Neill, Magee, Duerden (1999) J Clin Microbiol 37:2255-2261 Hall, Talbot, Stubbs, Duerden (2001) J Clin Microbiol 39:3555-3562 • Extract DNA from colonies • Amplify 16S ribosomal DNA by PCR • Digest DNA with restriction endonucleases – HaeIII and HpaII • Separate fragments© by in author agarose gel electrophoresis • Analyse banding patterns in GelCompar ESCMID• Compare with Online reference Lecture strain patterns Library HaeIII digest of 16S rDNA – various Actinomyces

ESCMIDL OnlineL LectureL LibraryL L = molecular size ladder 16S rRNA sequencing has become a mainstay of modern classification and identification

• ‘Universal’ for all • Specialist equipment prokaryotes and expertise needed • High copy numbers of • Time-consuming gene • Some spp. are not • Highly conserved and clearly differentiated highly variable regions • Other genes may be • Functional gene more discriminatory • Can indicate • Should be phylogenetic © by authorsupplemented with relationships simple tests ESCMID Online Lecture Library … and comparison of 16S rRNA sequences continues to indicate vast numbers of novel species / genera • Actinomyces cardiffensis Retrospective study of • Actinomyces ~500 Actinomyces vaccimaxillae isolates previously • Actinomyces oralis identified by conventional • Actinomyces nasicola phenotypic tests yielded • Actinomyces dentalis seven novel species and one novel genus / • Actinobaculum urinale species. © by •authorCorynebacterium atypicum

ESCMID Online Lecture• Varibaculum Library cambriense Consequently …

• Textbooks / ID schemes rapidly become obsolete • Most commercial ID databases have not kept pace with changes in nomenclature or recognition of novel species.

Some species not so clearly differentiated:

e.g. A. oris/viscosus/naeslundii - “naeslundii complex” – different serovars/subspecies

A.odontolyticus and A.meyerii – 99/98% homology - phenotypics still have© aby role! author ESCMID Online Lecture Library … and public-domain databases contain inaccuracies … Sometimes this is obvious

StrainESCMID SECO-Mt75m2 Online probably represents Lecture a novel Library Eggerthella species MALDI-TOF MS

• Very rapid • V.high-cost equipment • Low-cost consumables • Some spp. not clearly • Applicable to wide differentiated (as for range of organisms 16S rRNA seq.) • Direct detection e.g • Some organisms from blood culture difficult to extract • May detect mixed • Databases need further cultures development • Other applications© by author ESCMID Online Lecture Library MALDI-TOF MS

Now is the golden opportunity to develop robust and comprehensive databases based on large numbers of well-characterised strains....ENRIA!

Without these, identifications may be little better than those of current commercial systems. © by author ESCMID Online Lecture Library Eur J Clin Microbiol Infect Dis. 2012 Aug;31(8):1749-52. doi: 10.1007/s10096- 011-1496-3. Epub 2011 Dec 6. Comparison of phenotypic methods and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for the identification of aero-tolerant Actinomyces spp. isolated from soft-tissue infections. Ng LS1, Sim JH, Eng© by LC, authorMenon S, Tan TY. ESCMID Online Lecture Library Antimicrobial susceptibilities

• 'Classic' Actinomyces spp. are highly susceptible in vitro to penicillin and many other antimicrobials

• But in vivo, organisms are protected by abscess walls, microcolony formation

• Therefore aggressive therapy is required

 ? Empirical therapy for 'classic' species  May need broader spectrum for concomitant orgs  Disc diffusion unsuitable for slow-growing orgs  Etest / agar incorporation MICs recommended

Penicillin MICs (IU)  A. israelii, A. gerencseriae, A. naeslundii = ~0.016 – 0.064  A. odontolyticus = ~0.032 – 0.5  A. turicensis = ~0.016 – 0.25  A. urogenitalis = 0.5 – 1.5  A. turicensis, A. funkei,© by A. europaeus, author V. cambriense may be resistant to tetracycline and/or ESCMIDerythromycin/clinda Online Lecture Library Smith, Hall, Thakker & Gemmell (2005) JAC 56:407-9 Re-cap - Actinomyces • Normal flora, oral cavity etc. (common) • May cause actinomycosis (rare) • Plaque formation, dental caries • Involved in soft tissue infections • Difficult to isolate • Difficult to identify© by author • Susceptible to many antibiotics •ESCMIDDifficult to Onlinetreat Lecture Library Conclusions

• To establish clinical associations: thorough microbiology, realistic taxonomy and reliable ID are essential

• Much still to be discovered regarding pathogenicity / habitats

• Advances in technology (? microarrays) may make ID easier © by author • For the future: ? Direct detection from clinical specimens – ESCMID? which organisms Online to target Lecture Library Unusual Case: Disseminated infection due to A. meyeri: Case report & review.

Case: 47 yo M, alcohol abuser, smoker, had not brushed his teeth for 15 years! Dentogingival disease, numerous caries & stumps.

Presented with painful knee effusion; drained. 3/52 later; effusion recurred, febrile, lung abscess, osteomyelitis of tibia, multiple skin abscesses on trunk & extremities. A. meyeri & Actinobacillus actinomycetemcomitans isolated.

IV penicillin G 20 million U per day for 8 wks, then oral pen V 3 million U per day for 12 months. Multiple dental extractions. Surgical debridement & later© byre-construction author of knee joint.

Subcutaneous abscesses resolved in 2 wks. PulmonaryESCMID lesion regressed Online in 4 months. Lecture Library Apotheloz & Regamey Review summary: A. meyeri infections = rare but prob. under diagnosed. Dentogingival disease & alcoholism = major risk factors. Aspiration of mouth flora > pulmonary infection. Haematogenous spread to distant sites occurs particularly frequently with A. meyeri compared with other actinomycetes, often from pulmonary focus. Penicillin for several months© by author& surgical drainage = good prognosis.

ApothelozESCMID C & Regamey Online C (1996) Lecture Clin Infect Library Dis 22:621-5. Take-home message

Enjoy alcohol responsibly!

• Harriet Hughes (ARU Clinical Lead) • UK ARU team • Dr Val Hall • Dr Jon Brazier • Professor Brian© by Duerden author ESCMID Online Lecture Library