CMPT Clinical Bacteriology Program
Innovation, Education, Quality Assessment, Continual Improvement
www.cmpt.ca
November 2012
201 2
Challenge M123-5
Bone Swab: Co rynebacte rium amyco latum
HISTORY
MAIN EDUCATIONAL POINTS for M123-5
This challenge, sent to category A laboratories,
was a simulated bone swab sample obtained
from a 58 year old female diabetic patient with
osteomyelitis of the big toe.
1. Identify the potential significance of Corynebacterium species in
a normally sterile specimen.
Participants were expected to isolate and report
Corynebacterium amycolatum or Corynebacterium species and to perform and report results
of antimicrobial susceptibility testing for appropriate antibiotics.
CMPT QA
CMPT control yielded a pure growth of Corynebacterium amycolatum for 9 days.
SURVEY RESULTS
This survey achieved consensus for identification to the genus level of Corynebacterium species and is suitable for grading for the identification component. The reporting of antimicrobial susceptibility testing did not reach consensus
and, therefore, was not graded.
Reference Laboratories:
Identification Component. Eight laboratories
reported C. amycolatum, 5 laboratories reported Corynebacterium species ± refer or Coryne-
2. Identify the need for full identification and susceptibility testing of
Corynebacterium species in a normally sterile specimen.
3. Identify the significance of organisms that may normally be considered of low pathogenicity in the pathogenesis of diabetic foot
infections.
bacterium xerosis (correct to genus 12/14,
86%), 1 laboratory reported diphtheroids/
Corynebacterium as usual skin flora, and 1
laboratory did not submit a report. This component was considered suitable for grading.
Antimicrobial Susceptibility Component. Eleven
of 14 (79%) laboratories reported results of
antimicrobial susceptibility testing for penicillin
and vancomycin. All laboratories submitting
susceptibility results reported the isolate as
susceptible to both of these agents.
Grading
Maximum grade: 4
Reporting Corynebacterium
amycolatum was graded 4.
Reporting Corynebacterium
species was graded 4.
Reporting the wrong species
identification was downgraded to 3.
2 laboratories that did not perform susceptibility testing and one laboratory reported skin flora. Consensus was not reached for susceptibility testing and, therefore, was not graded.
Laboratories that provided
gram stain reaction only with
suggestive morphology received a grade of 3.
Participating Laboratories
Suggesting the isolate was
normal flora was graded 0.
Table 1. Identification results and grades assigned
Reported results
Corynebacterium amycolatum (25), Corynebacterium striatum/
amycolatum (4)
Corynebacterium species, +refer (2 added “not C. diphtheriae ± not
C.jeikeium”)
Corynebacterium xerosis
gram positive bacilli, resembling Corynebacterium species/coryneform
bacteria, diphtheroid – like, refer
diphtheroid/Corynebacterium species and called usual skin flora as per
Director
Corynebacterium amycolatum and Klebsiella pneumoniae
Total
Grade
29
4
26
4
1
3
12
3
1
0
1
0
mixed bacteria
1
0
no growth after 4 days, snnp
1
0
no report submitted / results submitted after due date
3
0
specimen not normally processed
7
ungraded
Total
82
Reporting other organism in
addition to the C. amycolatum was graded 0.
Not reporting results, reporting past the due date or using the wrong identifiers was
graded 0.
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CMPT Clinical Bacteriology Program November 2012
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Identification Component.
Component Reported identifications from participating laboratories are detailed in Table 1.
“diphtheroid” refers to the gram stain similarity
of these organisms to the most well-recognized
pathogen in the group, C. diphtheriae.
Twenty-nine laboratories identified the isolate
to species level as C. amycolatum or C. striatum/amycolatum, 63/82 (77%) of laboratories
identified the isolate to genus level as Corynebacterium species, as C. xerosis, or as some
variation of “gram-positive bacilli resembling
diphtheroids”, all of which indicated the isolate
would be referred for further identification. Two
laboratories in this latter group added the
Corynebacterium isolated was not C. diphtheriae. The 19 remaining laboratories reported a
variety of results as seen in Table 1.
Recent advances in molecular techniques have
led to many taxonomic changes in this group,
and many organisms formerly known as Corynebacterium species are now classified in many
other genera such as Turicella, Arthrobacter,
Brevibacterium, Dermabacter and others, in
addition to the Corynebacterium genus.
One laboratory reported “mixed bacteria”, and
1 laboratory reported C. amycolatum and
Klebsiella pneumoniae. These results indicate
contamination of the specimen at the point of
processing and these laboratories should review their procedures for sterile technique.
Susceptibility Testing Component. Tables 2 and
3 provide the details of the reports of antimicrobial susceptibility testing. All those labs reporting results indicated that the isolate was susceptible to both penicillin/amoxicillin and vancomycin. This component is ungraded since
consensus was not obtained. The tables are for
information purposes only.
TAXONOMY
The genus Corynebacterium is a diverse and
large group of microorganisms comprised of
irregularly shaped small gram positive bacilli
that do not form spores, which are commonly
referred to as “coryneform” bacteria or
“diphtheroids”.
Coryneform refers to the “club-shaped” morphology seen on Gram stain, and the term
Table 2. Susceptibility results - Penicillin
Reported results
There are over 80 species in the genus Corynebacterium, 50 of which are of clinical importance.1
ISOLATION AND IDENTIFICATION
The very typical club-shaped morphology on
Gram stain is only exhibited by true Corynebacterium species. Microscopically the organisms
are small gram positive, slightly curved, rods
without spores and with slightly wider ends,
which gives the club shaped appearance. Bacteria can appear as single cells, in pairs or palisading or as the so-called Chinese letter appearance.
Coryneform bacteria are not fastidious, do not
require special collection or transport conditions and will readily grow on 5% sheep blood
agar-based media. If deemed necessary, selective media are available for the recovery from
specimens that may have mixed normal flora.
Most will grow readily at 37ºC and will grow best
in a CO2 enriched atmosphere. Growth is generally visible within 48 hours.
When growth is examined in culture, colony
morphology, size, pigment, and hemolysis surrounding colonies can be helpful criteria leading to the identification of coryneform bacteria.
Characteristics of this group are too large and
varied to discuss in great detail in this survey.
Clinically relevant Corynebacterium species are
catalase positive, non-motile, and include both
Table 3. Susceptibility results - Vancomycin
Total
Reported results
Total
Susceptible
37
Susceptible
35
Amoxicillin Susceptible
1
Reported past due date
1
No report
1
no report
1
Reported past due date
1
n/a, refer, snnp
45
n/a, refer, snnp
42
Total
82
Total
82
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fermenters and non-fermenters. Most identification schema follow the outline of von Graevenitz and Funke based on catalase, fermentation or oxidation, motility, nitrate reduction,
urea hydrolysis, esculin hydrolysis, acid production from certain sugars, the CAMP reaction and
lipophilia.1
Commercial systems for identification and speciation of Corynebacterium species are used
widely, but the results of these systems should
be correlated with simple basic characteristics
such as gram stain and culture morphology and
often require supplemental tests.
C. amycolatum colonies are 1-2 mm in diameter after 24 hours incubation and are typically
dry, waxy, and grayish white with irregular edges.1 The strains demonstrate wide variability in
biochemical testing and can be misidentified
due to overlap with the reactions of other organisms.
Biochemically similar species include C. xerosis,
C. striatum, and C. minitissimum. In this challenge, 4 laboratories reported C. striatum/
amycolatum. These two species have different
colony morphologies: C. striatum is creamy and
moist, while C. amycolatum is dry and waxy.
One laboratory identified the isolate as C. xerosis, which will grow at 20ºC while C. amycolatum will not.
Definitive identification relies on 16S rDNA sequencing. There is limited data on the use of
the MALDI-TOF for the identification of this
group.
ANTIMICROBIAL SUSCEPTIBILITY
CLSI document M45-A2 “Methods for Antimicrobial Dilution and Disk Susceptibility Testing of
Infrequently Isolated or Fastidious Bacteria”
recommends penicillin, vancomycin, erythromycin and gentamicin for primary testing of
Corynebacterium species.2
Broth microdilution is the recommended method when testing is warranted, often requiring
blood supplemented media to ensure adequate
growth. Standardized MIC interpretative criteria
for Corynebacterium species is also provided in
this document.
Disk diffusion methods such as Kirby-Bauer are
not standardized and CLSI guidelines do not
give breakpoints for interpretation of disk diffusion methods. E-test methodology has been
reported in the literature to provide MIC results
comparable to those of broth dilution methods.3
Laboratories using Kirby-Bauer testing should
review their protocols to insure that MIC-based
methods are used or refer isolates to a reference laboratory for reliable results.
Antibiotic susceptibility pattern of Corynebacterium species is not predictable and isolates
from normally sterile sites should be tested.
Penicillin resistance is now common, and variable patterns of susceptibility are seen with
erythromycin, clindamycin, tetracycline and
ciprofloxacin.
The susceptibility of isolates to vancomycin is
predictable and this should be the drug of
choice until susceptibility patterns are known.
Corynebacterium species are also usually susceptible to linezolid.
Speciation and determination of antibiotic susceptibility patterns should be performed from
sterile sites as some species (C. jeikeium, C.
urealyticum, and C. amycolatum) may be resistant to many of the commonly used antibiotics including β-lactams, macrolides, and fluoroquinolones. 4,5
CLINICAL RELEVANCE
Corynebacterium species are part of the normal
flora, primarily of the skin and mucous membranes. With the exception of C. diphtheriae,
they are low virulence pathogens. However,
they can be associated with a variety of infections, particularly in patients whose defences
are compromised, either by disease, breaks in
barrier defenses, lowered immune status, or
medical interventions such as vascular access
lines or prosthetic devices.
Probably one of the hardest things to do in the
laboratory is to determine the clinical significance of coryneform bacteria isolated from
clinical specimens. The clinical relevance of
isolating these organisms in culture is strengthened when multiple specimens are positive for
the same Corynebacterium species and when
coryneform bacteria are seen in direct Gram
stains. 1,6
Further, coryneform bacteria warrant full identification if they are isolated from normally sterile
body sites from adequately collected specimens
if they are the predominant organisms. When
isolated from non-sterile sites with other normal
flora in similar quantities, coryneform bacteria
should be reported as normal flora.
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CMPT Clinical Bacteriology Program November 2012
201 2 M 123123 - 5
The patient history given in this survey indicated that the patient had diabetes and an osteomyelitis of the big toe.
Most of these infections are poly-microbial with
the most common organisms being gram positive cocci, particularly staphylococci. The Infectious Diseases Society of America (IDSA) has
recently published guidelines on diabetic foot
infections.8
It is recommended that physicians include the
possibility of osteomyelitis in the presence of
infected, deep, or extensive foot ulcers particularly if they overlie a bony prominence. These
experts believe that the most definitive way to
diagnose diabetic foot osteomyelitis is by both
bone culture and histopathology.
An appropriately collected bone sample, taking
steps to minimize contamination from potentially infected surrounding soft tissue, should
be sent for culture. A bone sample for culture
and histopathology is warranted when the diagnosis is not certain despite other investigations
and cultures from tissue specimens are not
definitive.
C. amycolatum is part of the normal human
skin flora and is the most frequently encountered Corynebacterium species in human clinical material. 1 This organism has been reported
in association with a wide variety of infections
including wounds, prosthetic and catheterassociated blood stream infections, urinary
tract infections, pneumonia, bacteremia and
endocarditis. 4,7
REFERENCES
1. Funke G, Bernard K. A. Coryneform grampositive rods. In: Versalovic ea, ed. Manual of
Clinical Microbiology. Vol 1. 10th ed. ed.
Washington, DC.: ASM; 2011:413.
2. Clinical Laboratory Standards Institute. Methods for Antimicrobial Dilution and Disk susceptibility Testing of Infrequently Isolated or
Fastidious Bacteria; Approved GuidelineSecond Edition. Wayne, PA.: CLSI; 2010;30:
M45-A2 CLSI Wayne, PA.
3. Martinez-Martinez L, Ortega MC, Suarez AI.
Comparison of Etest with broth microdilution
and disk diffusion for susceptibility testing of
coryneform
bacteria.
J
Clin
Microbiol
1995;33:1318-1321.
4. Reddy BS, Chaudhury A, Kalawat U, Jayaprada R, Reddy G, Ramana BV. Isolation, speciation, and antibiogram of clinically relevant
non-diphtherial
Corynebacteria
(Diphtheroids). Indian J Med Microbiol.
2012;30:52-57.
5. Funke G, Punter V, vonGraevenitz A. Antimicrobial susceptibility patterns of some recently established corynefrom bacteria. Antimicrob Agents Chemother 1996;40:2874-2878.
6. Bernard K. The Genus Corynebacterium and
Other Medically Relevant Coryneform-Like
Bacteria. J
Clin Microbiol. 2012;50:31523158.
7. Coyle MB, Lipsky BA. Coryneform bacteria in
infectious diseases: clinical and laboratoryoratory aspects. Clin Microbiol Rev. 1990;3:227
-246.
8. Lipsky BA, Berendt AR, Cornia PB, Pile JC,
Peters EJG, et al. 2012 Infectious Diseases
Society of America Clinical Practice Guideline
for the Diagnosis and Treatment of Diabetic
Foot Infections. Clin Infect Dis 2012;54:132173.
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