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The Wonderful World of

Weirdobacter species

Lars F. Westblade, Ph.D, D(ABMM)(E-mail: [email protected])

Weill Cornell Medicine

February 28th, 2017

No Financial Disclosures

P.A.C.E. #: 362-002-171

mailto:[email protected]

Objectives

• List key morphological and biochemical features of a

selection of microorganisms not commonly

encountered/recognized in the clinical microbiology

laboratory

• Describe their clinical significance

• Identify antimicrobial susceptibility patterns associated

with the microorganisms presented

2

What are Weirdobacter species and

Should You be Interested in Them?

• Organisms rarely encountered/recognized in the

clinical microbiology laboratory be at the ready!

• Can be difficult to identify using biochemical methods

• Provide opportunity for inane trivia!

3

Case 1: Color Me Bad

• 11-year-old male from Alabama presents to regional

community hospital with gluteal abscesses

• Had been standing in stagnant water

• PO clindamycin administered as outpatient

2 days

Richard et al., 2015 Am J Case Rep 16:740-444


• Within 24 h developed high fever and admitted to local

community hospital for surgical drainage of presumed

Staphylococcus aureus soft tissue infection

• IV clindamycin and ceftriaxone initiated

• Within 36 h of admission develops dyspnea/hypoxia

• Transferred to PICU at tertiary care medical center

5

Case 1: Color Me Bad • Patient urgently intubated for impending respiratory

failure progressive shock

• Physical examination revealed two large, indurated

abscesses on buttocks

• Multiple pustules on chest, abdomen, and extremities

6

7

• Developed acute respiratory distress syndrome

(ARDS)

• Placed on veno-arterial extracorporeal membrane

oxygenation (ECMO) to maximize cardiac output

• Rare Gram-negative rods (GNR) observed in wound

Gram stain

• Antimicrobial coverage: vancomycin, clindamycin,

nafcillin, ceftriaxone (changed to meropenem)



• Wound/blood cultures grew a GNR

• Grew on blood agar (BAP) violet pigmentation

Gram stain: medium sized GNR Violet-colored colonies on BAP

Image credit: https://www.cdc.gov/microbenet/8


• Chromobacterium violaceum

• Treated initially with meropenem, IV ciprofloxacin and

IV trimethoprim-sulfamethoxazole (SXT) – susceptible

• Discharged day 40 on ertapenem and PO SXT

• Auto-amputation 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

9

10

• Diagnosed with chronic granulomatous disease (CGD)

• c.75-76delGT mutation in NCF1 gene (encodes a

subunit of the phagocyte NADPH oxidase)

• Past medical history of submental abscess at 2 years

that grew Nocardia spp, supports diagnosis of CGD


C. violaceum Clinical Significance

• Inhabits soil/water in tropical/subtropical climates

• Clinical presentation (entry through skin [rarely oral]):- localized infection (contaminated wounds)

- Septicemia: multiple organ abscesses

• Endowed with adhesins, invasins, cytolytic proteins

• Associated with chronic granulomatous disease

(CGD)/glucose-6-phosphate dehydrogenase

deficiency high mortality rate

11

Chronic Granulomatous Disease

• Recurrent infection of lung, skin, lymph nodes, and

liver (granulomatous inflammation of affected organs)

• CGD infections caused by (catalase POS organisms):- Aspergillus spp

- Nocardia spp

- S. aureus

- (Pigmented) Gram-negatives: C. violaceum, Burkholderia

cepacia complex, Serratia marcescens, and Pseudomonas

aeruginosa

• Deficient NADPH oxidase complex results in impaired

production of reactive oxygen species (ROS) in

phagocytes, and ultimately a decrease in microbial

killing 12

13

Chronic Granulomatous Disease

Human NADPH oxidase complex

NADPH + 2O2 2O2- + NADP+ + H+

Human Superoxide dismutase

2H+ + 2O2- H202 + O2

Bacterial Catalase

2H2O2 2H2O + O2

C. violaceum Microbiology

• Motile GNR, glucose fermenter

• Facultative anaerobe

• Oxidase (OX) positive (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)

14

15Image credit: Selma Salter, NYPH-WCMC

C. violaceum:- OX, POS

- IND, V

- Lysine decarboxylase, NEG

- Violet pigmentation, V

- TCBS growth, NEG

Aeromonas spp:- OX, POS

- IND, POS

- Lysine decarboxylase, POS

(except, A. caviae complex)

- TCBS growth, NEG

Vibrio spp:- OX, POS

- IND, POS

- Violet pigmentation, NEG

- TCBS growth, POS


16

C. violaceum:- OX, POS

- IND, V

- Lysine decarboxylase, NEG

- Violet pigmentation, V

- TCBS growth, NEG

Aeromonas spp:- OX, POS

- IND, POS

- Lysine decarboxylase, POS

(except, A. caviae complex)

- TCBS growth, NEG

Vibrio spp:- OX, POS

- IND, POS

- Violet pigmentation, NEG

- TCBS growth, POS

Image credit: Selma Salter, NYPH-WCMC


Oxidase Activity of Pigmented Strains:

Ascending Paper Chromatography

Dhar and Johnson, 1973 J Clin Pathol 26:304-6

Slesak et al., 2009 Ann Clin Microbiol Antimicrob 8:24

Petri dish filled with 2-10 mL

oxidase reagent

Filter Paper

Organism

Schmear

Inset bottom of filter

paper in petri dish

17



Petri dish filled with 2-10 mL

oxidase reagent

Filter Paper

- + +Product of reaction

(indophenol blue)

“wicks” up paper

Oxidase Activity of Pigmented Strains:

Ascending Paper Chromatography

18

Testing Oxidase Activity of Pigmented

C. violaceum Strains



Schmear of organism

E. coli

-C. violaceum

+P. aeruginosa

+Distilled water (2-10 mL)

Schmear of organism

1% TMPD (oxidase reagent, 2-10 mL)

19

C. violaceum Antibiotic Susceptibility

McAuliffe et al., 2015 Am J Trop Med Hyg 92:605-10

• No established breakpoints for AST results

• High MIC values: polymyxins/some beta-lactams

• Low MIC values: fluoroquinolones, tetracyclines, SXT

20

Color Me Bad: Violacein

• Two molecules of L-tryptophan pyrrolidone scaffold

• Role in evading the immune system:- Neutralization of ROS

- Induce apoptosis of leukocytes (potential chemotherapy)

• Strong antimicrobial activity against bacteria/protozoa

Image credit: Lopes et al., 2009 Antimicrob Agents Chemother 53:2149-5221

Seeing Red: Serratia marcescens

Image credit: http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab2/isolated%20colonies.html

• Prodigiosin, red-colored pigment

(linear tripyrrole)

• Secondary metabolite produced

variably by S. marcescens

• Also produced by other Gram-

negative and -positive organisms,

including Serratia plymuthica and

Serratia rubidaea

• Antimicrobial and anti-tumor activity

• Very interesting history:- Blood on bread

- Operation Sea-Spray

22

Case 2

• 20-year-old female presented to local ED with fever,

hyotension, tachycardia (no significant past medical

hx)

• 6-8 week hx of intermittent vomiting, diarrhea, fever

• Received broad spectrum antibiotics, volume

resuscitation, vasoactive medications for septic shock

• CT revealed organomegaly

23

Case 2

• Transferred to our institution (PICU) intubated,

treated for hypotension

• Blood culture drawn day of admission positive with

GNRs within 24 h

• Treatment: pipercillin-tazobactam, vancomycin,

metronidazole, clindamycin, amphotericin B

• Abdominal distension emergent exploratory

laparotomy: viable, intact bowel, significant ascites,

liver congestion

24

Case 2

• Patient developed multi-organ dysfunction with ARDS

and expired

• Blood and lung cultures positive for GNR identified as

Burkholderia contaminans (member of the B. cepacia

complex [Bcc])

• Many necrotic foci in spleen/liver, innumerable micro

abscesses

25

Bcc Clinical Significance

• Members occasional opportunistic human pathogens

• Environmental organisms: soil/water (phytopathogens)

• Nosocomial pathogens, tolerance to disinfectants

• Associated with cystic fibrosis and CGD

Image credit: http://aces.nmsu.edu/pubs/_circulars/CR538/welcome.html

Sour Skin: B. cepacia Slippery Skin: Burkholderia gladioli

26

Cystic Fibrosis

Image credit: https://en.wikipedia.org/wiki/Cystic_fibrosis

• Autosomal recessive disease, afflicts

~60,000 individuals/worldwide

• Mutation of transmembrane protein,

CFTR (anion channel)

• Mutation of CFTR decreases its

activity accumulation of

dehydrated, physically altered

secretions in lung airways

• Altered secretions predispose

individuals to chronic endobronchial

infections inflammation and

permanent lung damage

27

• Motile GNR, aerobic glucose non-fermenters

• 18 members, including B. contaminans

• B. cenocepacia/B. multivorans most commonly

isolated from CF patients in US

Bcc Microbiology

B. cepacia:

Yellow colored colonies on BAPB. cepacia Gram stain:

medium sized GNR

Image credit: https://www.cdc.gov/microbenet/28

Bcc Microbiology/Antibiotic

Susceptibility• OX POS or V, glucose non-fermenter

• Pigmented:- some strains B. cepacia sensu stricto yellow-colored

- some stains B. cenocepacia brown-colored

• Resistant to polymyxins and aminoglycosides

• May only be susceptible to ceftazidime, levofloxacin,

meropenem, minocycline, or levofloxacin

• Our isolate susceptible to ceftazidime, levofloxacin,

meropenem, SXT (minocycline not tested) 29

Case 3: Look What the Dog Dragged In• 4-year-old mixed breed dog

• Rescue recently imported from Thailand

• Paralyzed hind legs (spinal injury), urinary incontinent

• 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

Image credit: http://www.handicappedpets.com/blog/30

Case 3: 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 (>2.0)

• Culture immediately moved to BSL-3 facility

Image credit: Cornell University, AHDC31

Case 3: 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!

32

B. pseudomallei Clinical Significance

• Agent of human/animal melioidosis (~165,000 cases

yr/worldwide)

• Endemic in SE Asia, tropical N Australia: 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

has been associated with clusters33


http://www.promedmail.org/34

• Acquired though inoculation, inhalation, aspiration and

ingestion (contaminated water sources)

• Presentation (incubation period, 2 days-many yrs):- acute, localized infection: ulcer, nodule or skin abscesses

- pulmonary infection (common): bronchitis, pneumonia, cavitary lesions

- acute, bloodstream infection

- chronic suppurative infection: joints, lymph nodes, liver, lung, spleen

• Risk factors: diabetes, renal insufficiency, cystic

fibrosis, alcohol excess (zoonotic transmission rare)

• Suspect in individuals with travel to endemic area who

present with fever of unknown origin or TB-like illness


35

B. pseudomallei Microbiology

Image credit: Manual of Clinical Microbiology, 11th Edition

• Small bipolar staining GNR, glucose non-fermenter

• Culture:- BAP: small, smooth, creamy in first 48 h further incubation,

wrinkled/crinkled colonies

- Ashdown agar: pinpoint colonies at 18 h purple, flat, dry and

wrinkled/crinkled colonies after 48 h incubation

Colony Gram stain Growth on BAP Growth on Ashdown agar

36

Wrinkled/Crinkled MicrobesTest B. pseudomallei

(GNR, bipolar

staining)

B. mallei

(Gram-negative

coccobacillus)

B. thailandensis Pseudomonas

stutzeri

Oxidase + V + +

Motility

(# of flagella)+

(≥2)

0 +

(≥2)

+

(1)

Growth on

MacConkey+ V + +

Growth at 42˚C + 0 + V

Wrinkled/crinkled

colonies (>48 h

incubation)

+ 0 + +

Arginine

dihydrolase+ + + 0

Arabinose

assimilation0 0 + Unknown

(to the presenter)

Polymyxin

susceptibilityR R R S

Abbreviations, + positive; 0, negative; V, variable; R, resistant; S, susceptible 37

B. pseudomallei Antibiotic Susceptibility• Intrinsically resistant to:- penicillins

- 1st and 2nd cephalosporins

- aminoglycosides

- macrolides

- polymyxins

• Resistant to aminoglycosides (B. mallei susceptible);

susceptible to amoxicillin-clavulanate (AMC) - unusual

• Treatment: - initial intensive therapy: ceftazidime or meropenem

- eradication therapy: SXT or AMC

• Postexposure treatment: SXT or AMC 38

B. pseudomallei Antibiotic Susceptibility

Image credit: Dr. David Dance, Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit

Amoxicillin-clavulanate

Colistin

Gentamicin

39

Potential for B. pseudomallei in the US?

Portacci et al., 2017 J Am Vet Med Assoc 250:153-940


Glass et al., 2006 Int J Syst Evol Microbiol 56:2171-641

Glass et al., 2006 Int J Syst Evol Microbiol 56:2171-6


42

Case 4: the Bite is Worse Than the Bark

• Previously healthy 22-mo-old boy presented to

community hospital ED day of a dog bite

• Wound irrigated, sutured, and prescribed amoxicillin

• Presented day later with purulent discharge from

eyelid. Sutures removed and prescribed ampicillin-

sulbactam

• Transferred to our hospital for care. Culture of the

wound performed

Yi et al., 2016 Pediatr Infect Dis J 35:214-643

Case 4: the Bite is Worse Than the Bark

• Sticky, tan-colored colonies noted after 48 h (in CO2)

– GNR

• MALDI-TOF MS (Bruker): Bergeyella zoohelcum (>2.0)

44Image credit: Selma Salter, NYPH-WCMC

B. zoohelcum Clinical Significance

• Normal oral/nasal flora cats and dogs

• Most human infections result of dog/cat bites

• Septicemia in elderly patients with skin infections (cat

slept on legs)

• Meningitis in child bitten on face/neck by dog

• Bacteremia in patient who ingested goat blood

45

B. zoohelcum Microbiology

• Very similar to Weeksella virosa

• OX/IND POS GNR, glucose non-fermenter

Test B. zoohelcum W. virosa

Colony

(color)Sticky

(tan-yellow)

Slimy

Oxidase + +

Indole + +

Pyrrolidonyl

arylamidase0 +

Urease + (rapid) 0

Polymyxin

susceptibilityR S

Abbreviations, + positive; 0, negative; V, variable; R, resistant; S, susceptible 46

B. zoohelcum Urease Activity

NEG B. zoohelcum

~5 minNEG B. zoohelcum

~15 min

47

B. zoohelcum Antibiotic Susceptibility

• Susceptible to penicillins (W. virosa too)

• Resistant to polymyxins

• Our isolate exhibited the following AST profile:- penicillin, 0.12 µg/mL

- amoxicillin, 0.12 µg/mL

• Our patient treated with PO amoxicillin-clavulanate and

recovered

48

Microbiology of Cat/Dog Bite Infections

Talan et al., 1999 New Eng J Med 340:85-92

• In US animal bites 300,000 ED

visits/yr (90% dog and cat bites)

• Occasional sequelae: meningitis,

endocarditis, septic arthritis, and septic

shock

• 107 patients: 50 dog bites/57 cat bites

• Polymicrobial infections:- aerobes/anaerobes from 56% of wounds

- aerobes alone from 36% of wounds

- anaerobes alone from 1%

- no growth 7%

• Pasteurella spp most commonly isolated

• Appropriate treatment: beta-lactam/beta-

lactamase inhibitor combination

49

Pasteurella multocida Gram stain:

Gram-negative small coccoid rod

Cat/Dog Bite Infections: Pasteurella spp

P. multocida on BAP

• Growth on BAP, chocolate agar, but not MacConkey

agar

• COIN: Catalase, OX, IND and Nitrate reduction POS

• Susceptible to penicillin

50Image credit: https://www.cdc.gov/microbenet/

51Image and case (and pun) credit: Stephen Jenkins, Weill Cornell Medicine

Puts a Twist on Things!

• Anaerobiospirillum succiniciproducens

• Bacteremia in a patient who kissed their

dog

• Anaerobic, long spiral GNR

• Corkscrew motility

• Associated with dog and cat feces

Case 5: It’s not Easy Being Green

52

• 30-year-old male sustained traumatic freshwater injury

jet-skiing in Missouri (immunocompetent)

• Cultures of adipose tissue grew green-colored

organism on Sabouraud dextrose agar

Westblade et al., 2015 New Eng J Med 372:982-4

Image credit: Brad Ford, University of Iowa

53


Image credit: Brad Ford, University of Iowa

10 µm

54

Colleagues,

We have isolated an organism we are pretty sure is Prototheca but colony morphology doesn't fit. MCM describes colonies as white and yeast-like. This colony is a green as can be on Sab Dex. Microscopically, it appears as an oblong rather than round algae, so I assume it is a P. zopfii. Patient had a water exposure. Does anyone know of a lab that could verify the identity?

Thanks

Bob

Bob Fader, Ph.D. D(ABMM)Section Chief, Microbiology/VirologyScott & White HealthcareTemple, TX 76508254-724-2714 (office)254-724-8776 (fax)

Meanwhile in Texas…

tel:254-724-2714

tel:254-724-8776

55


• Both Missouri and Texas isolates identified as

Desmodesmus armatus: chlorophyll-containing alga

• Both Missouri and Texas patients required surgical

debridement, no antifungals administered

• Both recovered without recurrence of D. armatus

infection

56

Human Protothecosis

• Protothecosis caused by infection with achlorophyllic

algae

• Two spp cause infections in human:- Prototheca wickerhamii (most common)

- Prototheca zopfii

• ~100 cases described in the literature:- cutaneous infection (66%)

- systemic infection (19%)

- olecranon bursitis (15%)

• Treatment: antifungals (amphotericin B) and surgery

57

Human Protothecosis

Lass-Flörl and Mayr, 2007 Clin Microbiol Rev 20:230-42

Hematoxylin and Eosin stain,

cutaneous protothecosis (1,000 ×) Wet mount, lactophenol cotton blue

Calcofluor white staining

58

Human Chlorellosis

• Chlorellosis caused by infection with chlorophyllic

algae

• Two organisms described in human infections:- Chlorella spp

- D. armatus

• A handful of cases described (4?, including ours)

• Treatment: surgery, antiseptic baths

59

Human Chlorellosis

Jones et al., 1983 Am J Clin Pathol 80:102-7

Image of lesion PAS-positive cytoplasmic granules

in granulomatous lesion (1,500 ×)

TEM of degenerated, collapsed algal cell

containing membranous profiles remnant

of a chloroplast, CP (28,440 ×)

MicrobeNet

https://www.cdc.gov/microbenet/60

61

16S rRNA gene

sequence search

Phenotypic search

MALDI-TOF MS search

The goal of MicrobeNet is to provide the same

information on many bacterial and fungal species

curated by the CDC, regardless of the

level of technology of the laboratory.

Slide courtesy John McQuiston: Special Bacteriology Reference Laboratory, CDC

MicrobeNet

https://microbenet.cdc.gov/

https://microbenet.cdc.gov/

AcknowledgementsEileen Burd and EUSM Clinical Microbiology Staff

Bart Currie, Menzies School of Health Research

David Dance, LOMWRU

Mike Dunne/Carey-Ann Burnham and BJH Clinical Microbiology Staff

Robert Fader, Baylor Scott & White Health

Brad Ford, University of Iowa

Steve Jenkins and NYP-WCMC Clinical Microbiology Staff

Bob Jerris and CHOA Clinical Microbiology Staff

Anil Thachil, Cornell University College of Veterinary Medicine

Jumi Yi, Emory University62

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