Cases in Clinical Microbiology

The case description on this page and its follow-up discussion presented
elsewhere in this issue is the 23rd in a series of articles presenting clinical
microbiology cases that will appear in this journal. Readers should study the case de-
scription below and formulate their own answers to the questions posed. After coming
up with a solution to the problem, turn to page 56 in this issue and read the Case Follow-
up and Discussion.This is followed by Questions for STEP Participants on page 59.
Joel E. Mortensen, PhD, MLT(AMT), Series Editor

C ase Description: A 20-year-old male with a history of spina bifida in the

lumbar region, type II diabetes mellitus, solitary left kidney, neurogenic
bladder and bowel, and chronic sacral decubiti presented to the emergency
department at Cincinnati Children’s Hospital Medical Center with fever,
nausea, and vomiting. On examination, there were multiple sacral ulcers, with
the largest stage 4 ulcer present on the left buttock and measuring 5cm in
diameter by 5cm deep with purulent drainage. A swab specimen of the wound
from the left buttock ulcer was initially sent for culture. Following processing
and incubation in the laboratory, this culture grew seven different colony-types
with no predominant type. A more representative specimen was requested. The
patient was subsequently taken to the operating room for debridement. Tissue
obtained during this procedure from the ulcer bed was sent for culture. The
specimen was cultured on 5% sheep blood agar (SBA) and chocolate (CHOC)
agar. The media were incubated at 35°C in 5% CO2 for 24-48 hours. Following
incubation, there was a predominant growth of many tiny grey colonies on
CHOC agar. These colonies were small and -hemolytic on SBA.(Fig.1).

40 April 2012 • Continuing Education Topics & Issues

Fig 1: SBA examined with transmitted light, tiny Fig. 2: Gram-stained smear of isolated colony from 24hr growth on SBA
-hemolytic colonies. Pictures by Chris Woods, 2011 showing pleomorphic Gram-positive bacilli. Pictures by Chris Woods, 2011

At this point, the major consideration was a possible Streptococcus spp. A Gram
stain, catalase test, and latex streptococcus grouping test to identify Lancefield
streptococcal groups A, B, C, D, F and G were performed. The microorganism,
however, was a pleomorphic small, v-shaped, branching Gram-positive bacillus
(Fig. 2), catalase-negative, and unreactive with the Lancefield streptococcal
grouping antibodies. The Vitek 2 bioMerieux system reported the
microorganism as an UNIDENTIFIED organism. In the meantime, the patient
was treated with piperacillin-tazobactam and daily wound dressing. He
promptly improved after 6 days of antibacterial therapy, remained afebrile with
improvement of nausea and vomiting, and continued antibiotics for an
additional week after discharge. Which bacteria should be considered at this
time and what should be done with the isolate at this point?

Continuing Education Topics & Issues • April 2012 41

Article 388 CASES IN CLINICAL MICROBIOLOGY
1 Clock Hour

Case Twenty Three:

Not Just in Animals
Oluyomi Asojo, Ashley Bowman, and Joel Mortensen

The mystery microorganism is a tiny, Gram posi-

EDITOR’S NOTE: BEFORE reading the Case tive rod that is strongly -hemolytic, catalase nega-
Follow-up and Discussion below, study the Case tive, ferments xylose and demonstrates no reaction
Description on page 40 of this issue, and formu- on the reverse CAMP test. What is your diagnosis?
late your own answers to the questions posed.
Background
Arcanobacterium pyogenes is a well known
Case Follow up pathogen in animals, causing a number of pyogenic
The considerations were Listeria, Corynebactri- infections in cattle and swine.1-6 Very few cases of in-
um, Erysipelothrix and Arcanobacterium species. The fection in humans due to A. pyogenes are reported in
isolate was catalase-negative, which rules out English literature, and they are almost always exclu-
Corynebacteria and Listeria species. The Gram-posi- sive to rural settings.1-10 A. pyogenes was initially
tive card of the Vitek 2 system can identify known as Corynebacterium pyogenes8 and later as
Erysipelothrix species11, which eliminated that genus. Actinomyces pyogenes. Based on 16S rRNA gene se-
At this point, a reverse CAMP test was performed to quences, it was assigned to the genus Arcanobacteri-
determine if the microorganism might be Arcanobac- um in the year 19977. It is classified as a member of
terium hemolyticum. A. hemolyticum typically inhibits the class, Actinobacteria; order, Actinomycetales;
the beta hemolysis of Staphylococcus aureus on blood family, Actinomycetaceae and genus Arcanobacteri-
agar, hence resulting in a positive reverse CAMP test um. It belongs to the lineage of Gram-positive bacte-
(Fig. 3). The patient's isolate neither inhibited nor en- ria with high guanine + cytosine content.12 It is gener-
hanced the -hemolysis of S. aureus and was not ally discussed under coryneform Gram-positive
A. hemolyticum. Additional biochemical tests were bacilli but it does not exhibit club shaped morphology
performed and are summarized in Table 1.: which is restricted to Corynebacterium spp. In Ar-
canobacteria spp, lysine is the amino acid in the cell
wall, but in the phylogenetically related Actinomyces
spp, lysine or ornithine can be found. Palmitic, oleic
and stearic acid are the main cellular fatty acids.12 The
Oluyomi Asojo, genus Arcanobacterium currently contains 6 species,
MD, Anatomic 3 of which have been recovered from human clinical
Pathology and specimens: A. hemolyticum, A. bernadiae, A. pyo-
Clinical Pathology,
genes. Unlike A. hemolyticum, which is a relatively
Cincinnati, OH;
Ashley Bowman, well known pathogen that causes pharyngitis in hu-
MT, Diagnostic mans, the role of A. pyogenes in human infections is
Testing Laboratory, not clearly established, and based on the limited num-
Dept. of Pathology; ber of case reports. This may partly be due to the fact
and Joel E.
Fig. 3: Reverse CAMP test: Zone of hemolysis around S. aureus that it can be misidentified as A. hemolyticum2. Some
Mortensen, PhD,
not inhibited. Picture by Chris Woods, 2011.
Director,
Diagnostic
Infectious Disease
Biochemical Catalase Glucose Maltose Xylose Lactose Nitrate Voges Esculin PYR
Testing Laboratory,
Dept. of Pathology, test reduction Proskauer hydrolysis
Cincinnati Result – + + + + – + – –
Children’s Hospital
PYR: L-pyrrolidonyl-␤- naphthylamide test , Negative test result: –, Positive test result: +. Biochemical tests performed at Focus Diagnostic Laboratory
Medical Center,
Cincinnati, OH Table 1: Biochemical reactions of the unidentified isolate

56 April 2012 • Continuing Education Topics & Issues

 Reference No. of Type of infection Underlying Contact Treatment Outcomes
cases illnesses history
[2] 7 Abdominal abscess, Cervical carcinoma, Not Specified Surgery, Penicillin, All recovered
Sigmoiditis/cystitis, Appendectomy, Sulfonamides,
Superficial abscesses, Diverticulitis, hloramphenicol,
Appendicitis, Cholelithiasis, Ampicillin or Amoxicillin
Cholecytitis, Otitis Cholesteatoma
media,
[5] 2 Subcutaneous lumbar Diabetes mellitus in Both tended hen Surgery, Ampicillin, Both recovered
bscess, Infected foot patient with foot ulcer house, lived in Ceftriaxone, Ofloxacin,
ulcer rural setting Metronidazole
Amoxicillin-clavulanate
[9] 1 Septic arthritis Diabetes mellitus Farmer Flucloxacillin. Penicillin, Recovered
Rifampicin, Amoxacillin
[3] 1 Chronic endocarditis Aortic stenosis Pigs & cattle Ceftriaxone, Gentamicin, Died
Amantadine, Cefotaxime,
Vancomycin, Ampicillin,
Penicillin
[1] 1 Acute endoarditis Diabetes Mellitus, None known Piperacillin-tazobctam, Died
Cancer, Cirrhosis Vancomycin, Penicillin
[4] 1 Bacteremia Colonic carcinoma Cattle & geese Amoxicillin, Recovered
Metronidazole
[10] 3 Infected foot ulcers Diabetes mellitus, Cattle Ceftriaxone, Ofloxacin, 2 recovered, 1
Hansen’s disease, Metronidazole, not determined
Scabies Ceftazidine, sulbactam,
amikacin. Amoxicillin-
Clavulanate
Our case 1 Infected sacral ulcer Diabetes mellitus None Known Piperacillin-tazobctam Recovered

Table 2: Summary of cases of A. pyogenes infection reported in English literature

authors believe that A. pyogenes infection in humans Laboratory Identification

is underreported, as it may be regarded as a coryne- A. pyogenes is an aerobic, asporogenous, pleo-
form contaminant.3,4 A. pyogenes, however, has morphic Gram-positive, non-motile bacillus. Due to
unique biochemical characteristics that can assist in its morphology, it may be mistakenly regarded as a
its laboratory identification. coryneform contaminant in specimens. A. pyogenes
grows well on SBA and can be isolated aerobically
Reservoir in CO2 enriched atmosphere when incubated at
A. pyogenes is a commensal in the upper respira- 35-37°C for 24-48hrs. A. pyogenes colonies are the
tory and genital tracts of domestic animals.3,6 It is a largest of all Arcanobacterium colonies, with diame-
well known opportunistic pathogen in these animals, ters up to 1mm after 48hrs of incubation. The
causing a number of pyogenic infections such as liver colonies are convex, white to grey and -hemolytic.
abscesses, arthritis, and pneumonia.1-3,5 It has not been Of all Arcanobacterium spp,. A. pyogenes shows the
described as part of the normal human flora2. sharpest zone of hemolysis. Polymysin is the protein
responsible for the -hemolysis, and is an important
Clinical Significance virulence factor in-vivo.12
A. pyogenes is a rare cause of infections in hu-
mans. Unlike A. hemolyticum, which produces a well Biochemical Characteristics
defined clinical infection in the upper respiratory A. pyogenes is catalase-negative and metabo-
tract2, confirmed cases of human A. pyogenes infec- lizes sugars by fermentation. It ferments glucose
tions are not site specific. Reported cases include soft with succinic and lactic acid as the main end prod-
tissue abscesses, otic infections, intraabdominal in- ucts. A. pyogenes is the only Arcanobacterium spp.
fections, cystitis, pneumonia, endocarditis and blood- of medical relevance that expresses -glu-
stream infections, foot ulcers, and others [Table 2]. A curonidase and ferments xylose. A. pyogenes has
common factor in majority of cases is the presence the ability to hydrolyze gelatin and reacts with an-
of underlying illnesses, such as cancer and diabetes tisera against Lancefield group G streptococci re-
mellitus, as seen in our index case. Infections are sulting in possible misidentification as Streptococ-
most commonly reported in people living in rural cus.2 The ability to produce -glucuronidase,
areas, who have close contact with animals. Table 2 hydrolyze gelatin, ferment xylose and produce a
summarizes cases of A. pyogenes infections in hu- negative reverse CAMP test differentiates A. pyo-
mans reported in English literature. genes from A. hemolyticum. The 3 medically rele-

Continuing Education Topics & Issues • April 2012 57

 vant Arcanobacteria are correctly identified by the 3. Reddy I, Ferguson DA Jr, Sarubbi FA. Endocarditis due to
API Coryne database system (API Coryne 1 sensi- Actinomyces pyogenes. Clinical Infectious Disease Decem-
ber 1997. 25(6):1467-7.
tivity 56-85%, API Coryne 2 sensitivity close to 4. M. Barnham Actinomyces pyogenes bacteraemia in a patient
100%). This commercial test system uses a combi- with carcinoma of the colon. Journal of Infection November
nation of standard biochemical tests and fermenta- 1988. Vol:17, Issue 3, 231-234.
tion tests. Definitive identification is best achieved 5. Drancourt M, Oules O, Bouche V, Peloux Y. Two cases of
by molecular methods using 16SrRNA-targeted Actinomyces pyogenes infection in humans. Eur J Clin Mi-
crobiol Infect Disease. January 1993.12 (1): 55-7
PCR amplification, sequencing and editing. Ampli- 6. Levy CE, Pedro RJ, Von Nowakonski A, Holanda LM, Brocchi
fied products are then compared to sequences avail- M, Ramo MC. Arcanobacterium pyogenes sepsis in farmer,
able in the NCBI Genbank bacterial DNA database. Brazil. Emerg Infect Dis. July 2009. 15 (7): 1131-2.
7. Cristina P Ramos, Geoff Foster, Mattew D. Collins. Phyloge-
Antimicrobial Susceptibilities netic Analysis of the Genus Actinomyces Based on 16S rRNA
Gene Sequences: Description of Arcanobacterium phocae sp.
Isolates from infected animals are susceptibile to nov., Arcanobacterium bernardiae comb. nov., and Arcanobac-
-lactams, gentamicin, macrolides, vancomycin, terium pyogenes comb. nov. Int J Syst Bacteriol 1997. 47; 46-
linezolid and rifampin and are resistant to TMP- 53.
SMX, streptomycin and tetracycline1. Susceptibility 8. T E Na'Was, D G Hollis, C W Moss, and R E Weaver. Com-
standards in human infections are not available; how- parison of biochemical, morphologic, and chemical character-
istics of Centers for Disease Control fermentative coryneform
ever, cases reported show a similar susceptibility pat- groups 1, 2, and A-4. J Clin Microbiol. August 1987. 25(8):
tern as seen in animal infections. 1354-1358.
9. Nicholson P, Kiely P, Street J Mahalingum K. Septic arthritis
Conclusion due to Actinomyces pyogenes. Injury October 1998. 29(8):
A. pyogenes is a common zoonotic pathogen that 640-2.
10. Kavitha K, Latha R, Udayashankar C, Jayanthi K, Oudea-
can cause infections in humans. Due to morphology coumar P. Three cases of Arcanobacterium pyogenes associ-
on Gram stain, it may be mistaken for a coryneform ated soft tissue infection. J Med Microbiology June 2010.
contaminant. It can also be confused with Strepto- 59(6): 739-9.
coccus due to reaction with Lancefield group G anti- 11. Microbial identification using the bioMerieux Vitek 2 system.
sera. Human infections may be more common than David H. Pincus. bioMerieux, Inc. Hazelwood, MO,USA.
12. Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie
reported. Its characteristic biochemical properties Louise Landry, Michael A. Pfaller. Manual of Clinical Micro-
can assist in identification and differentiate it from biology, Vol.1, 9th edition, 2007. ASM Press Washington, DC.
A. hemolyticum.
Acknowledgments
References Richard Porschen, PhD, ABMM, Scientific Di-
1. M. Plamondon, G. Martinez, L. Raynal and L. Valiquette. A
rector, Focus Diagnostics, Inc. Cypress, California.
fatal case of Arcanobacterium pyogenes endocarditis in a man
with no identified animal contact: cas report and review of lit- Christopher Woods - Imaging Specialist. Cincin-
erature. European Journal of Clnical Microbiology & Infec- nati Children's Hospital Medical Center. Cincinnati,
tious Diseases. 2007.Vol:26, No 9. 663-666. Ohio
2. Bente Gahrn-Hansen, Wilhelm Frederiksen. Human infections
with Actinomyces pyogenes (Corynebacterium pyogenes). Di-
agnostic Microbiology and Infectious Disease. May-June
1992. Vol:15, Issue 4, 349-354.

58 April 2012 • Continuing Education Topics & Issues

Questions for STEP Participants 388
Article
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1. Arcanobacterium pyogenes is primarily 6. Which of the following has a positive reverse

pathogenic in which of the followng? CAMP test?
A. humans A. A. pyogenes
B. plants B. A. pluranimalium
C. animals C. A. hemolyticum
D. fish D. A. bernadiae

2. Colonies of Arcanobacterium pyogenes on 7. All of the following are true concerning

sheep blood agar are most commonly exhibit A. pyogenes except which of the following?
which of the following? A. It is part of the normal human flora.
A. a hemolytic B. It may be disregarded as a coryneform
B.  hemolytic contaminant.
C. y hemolytic C. Most infections are associated with
D. None of the above underlying illness.
D. It has unique biochemical characteristics
3. The following have been associated with that can assist in laboratory identification.
Arcanobacterium pyogenes infection except
which of the following? 8. Biochemical properties of A. pyogenes
A. Exposure to pigs and cattle include which of the following?
B. Residing in a rural environment A. Xylose fermentation
C. Tending hen houses B. Hydrolyzes gelatin
D. Intravenous drug abuse C. -glucuronidase activity
D. All of the above
4. All of the following Arcanobacterium spp.
have been recovered from human clinical 9. A. pyogenes
specimens except which of the following? A. Reacts with antisera against Lancefield
A. A. pyogenes group G streptococci
B. A. pluranimalium B. Colonies on sheep blood agar range from
C. A. bernadiae <0.5 to 1mm, even though they are the
D. A. hemolyticum largest of all Arcanobacterium colonies
C. A and B
5. Based on Gram-stain morphology, which of D. None of the above
the following organisms appear similar and
can be confused with Arcanobacterium 10. A. pyogenes infections in humans:
pyogenes? A. Have a well defined clinical pattern
A. Listeria spp. B. Have been reported in patients with no
B. Erysipelothrix spp. known exposure to animals
C. Streptococcus spp. C. Occur mainly in the upper respiratory tract
D. A and B D. Are commonly encountered in clinical and
laboratory practice

Continuing Education Topics & Issues • April 2012 59