10/11/19 Case Studies Lars F. Westblade, Ph.D, D(ABMM) (E-mail: [email protected]) Weill Cornell Medicine SCASM, San Diego October 26, 2019 1 1 The Wonderful World of Weirdobacter weirdii species! Lars F. Westblade, Ph.D, D(ABMM) (E-mail: [email protected]) Weill Cornell Medicine SCASM, San Diego October 26, 2019 2 2 1 10/11/19 Disclosures • Grant/Research suPPort: - Accelerate Diagnostics, Inc. - BioFire Diagnostics, LLC - Hardy Diagnostics • Advisory Board: - Roche Molecular Systems, Inc. 3 3 Objectives • List diagnostic features of microorganisms not commonly encountered or recognized in the clinical microbiology laboratory • Describe their significance and clinical Presentation • Discuss their key antimicrobial suscePtibility traits 4 4 2 10/11/19 What are Weirdobacter weirdii Species? • Organisms rarely encountered or recognized in the clinical microbiology laboratory à be at the ready! • Can be difficult to identify using biochemical methods • Provide oPPortunity for inane trivia! 5 5 Case 1: Color Me Bad • 11-year-old male from Alabama Presents to regional community hospital with gluteal abscess • Had been standing in stagnant water • Oral (PO) clindamycin administered as outPatient 2 days Lesions ~2 cm in diameter 6 Richard et al., 2015 Am J Case Rep 16:740-4 6 3 10/11/19 Case 1: Color Me Bad • Within 24 h develoPed fever and admitted to local community hospital for surgical drainage of Presumed Staphylococcus aureus soft tissue infection • Intravenous (IV) clindamycin and ceftriaxone initiated • Within 36 h of admission develoPs dyspnea/hypoxia • Transferred to PICU at tertiary care medical center 7 7 Case 1: Color Me Bad • Patient intubated for imPending respiratory failure and Progressive shock • Physical examination revealed two large, indurated abscesses on buttocks • MultiPle Pustules on chest, abdomen, and extremities: concern for staPhylococcal infection or ecthyma 8 Richard et al., 2015 Am J Case Rep 16:740-4 8 4 10/11/19 Case 1: Color Me Bad • DeveloPed acute respiratory distress syndrome • Placed on venoarterial extracorPoreal membrane oxygenation (ECMO) to maximize cardiac outPut • Antimicrobial coverage: vancomycin, clindamycin, nafcillin (for Presumed staPhylococcal infection) • Rare Gram-negative rods (GNR) observed in wound Gram stain • Ceftriaxone added based on wound Gram stain result; later changed to meroPenem because of Patient’s decomPensation 9 9 Case 1: Color Me Bad • Wound/blood cultures grew a GNR • Grew on blood agar (BAP) à violet pigmentation Gram stain: medium sized GNR Violet-colored colonies on BAP 10 Image credit: https://www.cdc.gov/microbenet/ 10 5 10/11/19 Case 1: Color Me Bad • Chromobacterium violaceum • Treated with meroPenem, IV ciProfloxacin and IV trimethoPrim- sulfamethoxazole (SXT) – all low MIC values (theraPy Paired to meroPenem and ciProfloxacin once antibiotic suscePtibility data available) • Discharged on day 40 on ertapenem (?) and PO SXT • After ~4 mo of theraPy, ESR mildly elevated, continued on PO ciProfloxacin and SXT for a total of 6 mo theraPy • DeveloPed autoamputation of distal end of right thumb and several toes, required skin graft of dorsum of right foot • Returned to school 4 mo after initial illness, no other Permanent sequelae 11 11 Case 1: Color Me Bad • Diagnosed with chronic granulomatous disease (CGD) • c.75-76delGT mutation in NCF1 gene (NCF1 encodes a subunit of the Phagocyte NADPH oxidase) • Past medical history of submental abscess at 2 yrs that grew Nocardia spP, suPPorts diagnosis of CGD 12 12 6 10/11/19 C. violaceum: Clinical Significance • Inhabits soil/water in troPical/subtroPical climates • Clinical Presentation (entry through skin [rarely oral]): - Localized infection (wound infections) - Disseminated infection, sePsis • Endowed with numerous virulence factors: adhesins, invasins, cytolytic proteins • Associated with CGD/glucose-6-Phosphate dehydrogenase deficiency à high mortality rate 13 13 Chronic Granulomatous Disease • An inherited immunodeficiency disorder: inability of Phagocytes to kill microbes they have ingested (usually discovered early in childhood) • Recurrent infection (every 3 to 4 yrs) of lung, skin, lymPh nodes, and liver (granulomatous inflammation of affected organs) • CGD infections caused by (catalase POS [environmental] organisms): - Aspergillus spP - Nocardia spP - S. aureus - (Pigmented) Gram-negatives: C. violaceum, Burkholderia cepacia comPlex, Serratia marcescens, and Pseudomonas aeruginosa • Defective enzyme in Phagocytes - NADPH oxidase complex - results in imPaired Production of reactive oxygen species (ROS), in Phagocytes • ROS kill microbes à thus lower levels of ROS leads to decreased microbial killing 14 14 7 10/11/19 Relationship between CGD and Catalase- Positive Organisms Human NADPH oxidase complex - + + NADPH + 2O2 à 2O2 + NADP + H Human Superoxide dismutase + - 2H + 2O2 à H2O2 + O2 Bacterial Catalase 2H2O2 à 2H2O + O2 15 15 Relationship between CGD and C. violaceum in the SE USA 16 Macher et al., 1982 Ann Intern Med 97:51-5 16 8 10/11/19 C. violaceum: Microbiology • Motile GNR, glucose-fermenter (non-lactose-fermenter) • Facultative anaerobe • Cultures have almond-like odor (ammonium cyanide) • Pigmented (~90%) and non-Pigmented (~10%) strains • Oxidase (OX) POS/indole (IND) variable (V) - Non-Pigmented strains typically IND POS • Non-Pigmented strains can be confused with Aeromonas spP and Vibrio spP (automated systems can misidentify as Burkholderia pseudomallei) 17 17 C. violaceum: Microbiology Vibrio spp: Aeromonas spp: - OX, POS - OX, POS - IND, POS - IND, POS - Lysine decarboxylase, POS - Violet Pigmentation, NEG (excePt, A. caviae comPlex) - TCBS growth, POS - TCBS growth, NEG C. violaceum: - OX, POS - IND, V - Lysine decarboxylase, NEG - Violet Pigmentation, V - Almond-like odor - TCBS growth, NEG 18 Image credit: Selma Salter, Stamford Health 18 9 10/11/19 C. violaceum: Microbiology Aeromonas spp: Vibrio spp: - OX, POS - OX, POS - IND, POS - IND, POS - Lysine decarboxylase, POS - Violet Pigmentation, NEG (excePt, A. caviae comPlex) - TCBS growth, POS - TCBS growth, NEG C. violaceum: - OX, POS - IND, V - Lysine decarboxylase, NEG - Violet Pigmentation, V - Almond-like odor - TCBS growth, NEG 19 Image credit: Selma Salter, Stamford Health 19 Oxidase Activity of Pigmented Strains: Ascending Paper Chromatography Inset bottom of filter Filter PaPer paper in petri dish Organism Schmear Petri dish filled with 2-10 mL oxidase reagent (TMPD) Dhar and Johnson, 1973 J Clin Pathol 26:304-6 20 Slesak et al., 2009 Ann Clin Microbiol Antimicrob 8:24 20 10 10/11/19 Oxidase Activity of Pigmented Strains: Ascending Paper Chromatography - + + Product of reaction (indoPhenol blue) Filter PaPer “wicks” uP PaPer Petri dish filled with 2-10 mL oxidase reagent (TMPD) Dhar and Johnson, 1973 J Clin Pathol 26:304-6 21 Slesak et al., 2009 Ann Clin Microbiol Antimicrob 8:24 21 Testing Oxidase Activity of Pigmented C. violaceum Strains E. coli C. violaceum P. aeruginosa - + + Distilled water (2-10 mL) Schmear of organism 1% TMPD (oxidase reagent, 2-10 mL) Schmear of organism Dhar and Johnson, 1973 J Clin Pathol 26:304-6 22 Slesak et al., 2009 Ann Clin Microbiol Antimicrob 8:24 22 11 10/11/19 C. violaceum: Antibiotic Susceptibility • No established breakpoints for AST results • High MIC values: Polymyxins/some b-lactams • Low MIC values: fluoroquinolones, tetracyclines, SXT 23 McAuliffe et al., 2015 Am J Trop Med Hyg 92:605-10 23 Color Me Bad: Violacein • Ethanol-soluble violet Pigment • Two molecules of L-tryptoPhan à Pyrrolidone scaffold • Role in evading the immune system: - Neutralization of ROS - Induce aPoPtosis of leukocytes (Potential chemotheraPy) • Strong antimicrobial activity against bacteria/Protozoa 24 Image credit: LoPes et al., 2009 Antimicrob Agents Chemother 53:2149-52 24 12 10/11/19 Case 2: Look What the Dog Dragged In • 4-year-old dog (mixed breed) • Rescue recently imPorted from Thailand • Paralyzed hind legs (spinal injury), urinary incontinence • Urine culture requested because of incontinence Case Courtesy of K. Deriziotis, J. Moody, D. Peck, R. Franklin-Guild, and A. Thachil College of Veterinary Medicine, Animal Health Diagnostic Center, Cornell University 25 Image credit: httP://www.handicaPPedPets.com/blog/ 25 Case 2: Look What the Dog Dragged In • Urine Culture: - 24 h: >105 PinPoint colonies on BAP and eosin methylene blue agar (EMB) - 48 h: mucoid colonies on BAP and EMB (NLF) • MALDI-TOF MS (Bruker), Burkholderia thailandensis (≥2.00) • Culture immediately moved to BSL-3 facility 26 Image credit: Cornell University, AHDC 26 13 10/11/19 Case 2: Look What the Dog Dragged In • “PresumPtive B. pseudomallei” NYS DOH PHL Biodefense Laboratory (determined by PCR) • Confirmed as an atypical strain of B. pseudomallei at CDC • Atypical characteristics: - Beta-hemolytic on BAP at 48 h (sometimes observed around areas of confluent growth, not around individual colonies) - Catalase NEG – unusual! • Tier 1 select agent; Federal Select Agent Program 27 httPs://www.selectagents.gov/ohP-app1.html 27 B. pseudomallei: Clinical Significance • Agent of human/animal melioidosis (~165,000 cases yr/worldwide) • Endemic in SE Asia, N Australia, India, China, Caribbean (Puerto Rico) - troPical areas: case fatality rate estimated as high as 50%! • Environmental organism, resident in soil/water • Infection is seasonal, uP to 85% of cases during monsoon wet season • Severe weather events/environmental disturbances have been associated with clusters (e.g., Asian tsunami 2004) 28 28 14 10/11/19 B. pseudomallei: