Actinomyces in Human Infections

Actinomyces in human infections

Trefor Morris, Lead Scientist, UKARU Cardiff, Wales (not England!)

Fastest growing capital city in Europe...

University city with its own half marathon...

Trefor Morris, Actinomyces in human infections 2016 UK Anaerobe Reference Unit

• Serves the entire UK • One of a select few world-wide • Remit of the ARU includes: 1. Identification & susceptibility testing 2. C. difficile ribotyping and surveillance (Wales) 3. Clinical & technical advice 4. Maintenance of a reference collection of clinical isolates c.40,000© by author 5. Training ESCMID6. R&D, product Online evaluations Lecture Library

Trefor Morris, Actinomyces in human infections 2016 What’s coming up...

1) History of Actinomyces 2) Microbiology basics 3) Laboratory processes 4) Taxonomy & recent changes 5) Microbiota 6) Spectrum of disease 7) Classical clinical associations 8) Newer clinical ©associations by author 9) Future ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 History (technical)...

“Adopted Anaerobes”...

Historically identified by “Anaerobe Enthusiasts” due to technology and slow growth (5-10days)

ARU: methods developed (ARDRA) now overtaken by 16S © by author

ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 History - Actinomycosis

Actinomycosis in humans - Wolf & Israel, 1891

‘LumpyESCMID jaw’ in Onlinecattle - Bollinger Lecture / Harz Library 1879

Trefor Morris, Actinomyces in human infections 2016 Microbiology (basics)

• Non-sporing gram positive rods • Facultative anaerobes... • Metronidazole resistant • Catalase variable • Indole negative • ~40 species, ~20© by affecting author humans •Some slow growing (5-10 days) ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 Microbiology - colonial appearance

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

Trefor Morris, Actinomyces in human infections 2016 A. graevenitzii A. cardiffensis

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

Red under UV light

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 Laboratory processes Specimen problems: • If disease is not obvious it may be difficult to get specimen

• Diagnosis = Ca. Tissue in formalin = histology only

• Pus drained, but only swab sent to lab.

• Dry swab = worst case © by author • Unsuitable: oral swab, sputum, vaginal swab • LackESCMID of clinical Online info. = may Lecture not culture Library for Actinos

Trefor Morris, Actinomyces in human infections 2016 Good specimens

Cerebral abscess pus

Direct Gram's stain of crushed granules

CultureESCMID = No growth Online @ 10 days. Lecture Prior antibiotics, Library old specimen .

Trefor Morris, Actinomyces in human infections 2016 Culture for Actinomyces 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 5-10 days

Infections usually polymicrobial and other organisms grow faster  broth ©enrichment by author is rarely useful

MayESCMID only grow anaerobically Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 Problems - Identification • Slow-growing / gritty colonies

• New species – not in textbooks / commercial databases

• Lack of familiarity / experience

• Historical kits based on phenotypics unreliable © by author • Modern methods (Maldi-Tof, 16S) improved ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 … 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 •authorCorynebacte rium atypicum

ESCMID Online Lecture• Varibaculum Library cambriense

Trefor Morris, Actinomyces in human infections 2016 A good trace and clear-cut identification:

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 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 LC, Menon S, Tan TY.

Actinomyces ARU data (unpublished)© by author correct presented at ECCMID incorrect 2013.ESCMID Direct spot Onlineonly. Lecture Libraryno reliable ID

Trefor Morris, Actinomyces in human infections 2016 Antimicrobial susceptibilities

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

 Others more resistant e.g. A. turicensis, A. funkei, A. europaeus, V. cambriense may be resistant to tetracycline and/or erythromycin/clinda • But in vivo, organisms are protected by abscess walls, microcolony© formation by author • Therefore aggressive therapy is required

• ESCMIDResistant to metronidazole!Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 Susceptibility testing

 ? 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© – by1.5 author

ESCMIDSmith, Hall, OnlineThakker & GemmellLecture (2005) Library JAC 56:407-9

Trefor Morris, Actinomyces in human infections 2016 Taxonomy

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 by that author most bacteriologists deal with • Species - •ESCMIDSub-species Online- Lecture Library

Trefor Morris, Actinomyces in human infections 2016 Classification of Actinomyces

• Phylum = Actinobacteria • Class = Actinobacteria • Order = Actinomycetales • Family = Actinomycetaceae • Genus = Actinomyces © by author ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 1990 2001 human and animal species • A. hyovaginalis • A. bowdenii • A. bovis (type strain) • 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

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 …but closely related to…

• Arcanobacterium • Actinobaculum • Varibaculum • Mobiluncus © by author ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 Taxonomic troubles!

• Some species re-assigned to different genera e.g. Actinobaculum suis, Truperrella bernardiae

Actinobaculum schaalii & urinale then re-assigned to Actinotingum...

A. naeslundii/’viscosus© by’ group author actually 4 distinct species – A.naselundii, A.oris, A.johnsonii ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 Microbiota of mammals

• Oral cavity (tongue, plaque, periodontal)

• Gastro-intestinal tract

• Genito-urinary tract (small numbers)

• Lipid rich areas of skin

Trefor Morris, Actinomyces in human infections 2016

Pathogenicity of Actinomyces species

Actinomyces spp. initiate a 'foreign body' response from the immune system Dense filamentous microcolonies (eosinophilic) Surrounded by basophilic material (bacterial and host) Granulomas / cavitations / sinuses develop Bacteria protected from further host responses Protected from antimicrobial© by author activity SimilarESCMID to TB interaction Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 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 (A.oris and A.naeslundii) • Significant in dental caries (A.israelii and A.gerencseriae© by author) • Periodontal diseases • ESCMIDRoot canal infections Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 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 .

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 Pelvic actinomyces / IUCDs

• 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

Trefor Morris, Actinomyces in human infections 2016 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

Trefor Morris, Actinomyces in human infections 2016 Broad clinical associations... • 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

ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 More specific clinical associations...

Actinomyces graevenitzii has predilection for respiratory sites

Actinomyces meyerii – Brain Abscesses and severe CNS infections

Actinomyces turicensis – most common below waist

Varibaculum cambriense – soft tissue infections

Trefor Morris, Actinomyces in human infections 2016 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 • relapse / treatment© byfailure author = common • May be eventually fatal if not treated ESCMID Online Lecture Library

Trefor Morris, Actinomyces in human infections 2016 Conclusions & future considerations • To establish clinical associations: thorough microbiology,accurate taxonomy and reliable ID are essential

• Advances in technology makes this easier (Maldi-Tof)

• Much still to be discovered regarding pathogenicity / habitats

?Whole genome sequencing for analysis of pathogenicity?

Trefor Morris, Actinomyces in human infections 2016 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 C & Regamey C (1996) Clin Infect Dis 22:621-5.

Trefor Morris, Actinomyces in human infections 2016 Take-home message

Enjoy alcohol responsibly!

Trefor Morris, Actinomyces in human infections 2016 Acknowledgments • Dr Harriet Hughes (ARU Clinical Lead) • Val Hall • UK ARU laboratory team • ESGAI

References • Hall V (2008). Actinomyces – gathering evidence of human colonization and infection. Anaerobe 14:1-7.© by author

• Kononen & Wade – Actinomyces and related organisms in human infections ESCMID Clinical Microbiology OnlineReviews. April 2015. Lecture Library

Trefor Morris, Actinomyces in human infections 2016