Educational Workshop
EW04: Practical approach to diagnose mixed anaerobic
infections in "real time“
Arranged with the ESCMID Study Group for Anaerobic
Infections (ESGAI)
Convenor:
Elisabeth Nagy (Szeged, HU)
Faculty:
Diane .M. Citron (Culver City, US)
Ulrik Stenz Justesen (Odense, DK)
Georg Conrads (Aachen, DE)
Elisabeth Nagy (Szeged, HU)
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Classical methods for identification
of anaerobes from mixed
infections: do we still need them?
Diane M. Citron
 R.M. Alden Research Lab
 Culver City, California

Survey of USA Hospital
Labs (2006-2007)
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150 hospital laboratories (200-1000 beds);
85% responded to questionnaire
85% processed anaerobic cultures
15% sent to reference lab
100% used selective and differential media
for isolation (in addition to blood agar)
30% use identification disks
66% use preformed enzyme kits
30% use other biochemical tests
4% GLC
0% used molecular methods
Goldstein EJ et al, 2008, Anaerobe 14:68-72
Practical considerations


Few commercial molecular kits
available for routine anaerobic
specimens except in specialized
areas (C. difficile toxin)
Molecular methods mostly limited to
in-house preparations in large labs or
research institutions
1
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Incidence of anaerobes
in clinical specimens

B. fragilis group
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B. fragilis
B. thetaiotaomicron
Other Bf group
Prevotella-Porphyromonas etc.
Fusobacterium
Anaerobic cocci
Clostridium
Non-sporeforming GPR
35%
(40-50%)
(20%)
(20-40%)
15-20%
5-10%
25-30%
8-15%
5-10%
What classical methods are
being used?
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Selective and differential agar media to
culture anaerobes
Rapid tests such as catalase, spot indole,
urease, nitrate
Identification kits – RapID ANA II, Rapid
ID 32a and others
Tube biochemicals
GLC for short and long chain fatty acids
Selective and differential media

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Bacteroides bile-esculingentamicin agar (BBE)
Rapid presumptive ID of B.
fragilis group, Alistipes, and
Bilophila.
Some strains of Fusobacterium
mortiferum and F. varium may
grow. (bile resistant)
Enterococci highly resistant to
gentamicin will grow
2
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Selective and differential media
Laked blood with kanamycin and
vancomycin brucella agar (LKV)
 Inhibits gram-positives and enterics
 Grows Bacteroides, Prevotella,
Alistipes some fusobacteria(and Van-R
Clost. innocuum!)
 Enhances pigmentation of
Prev. melaninogenica group

Growth on Selective and
differential media
EYA-Fusobacterium
necrophorum (FEA) PEA Porphyromonas
LKV-BBE B. fragilis
Prevotella
species
F. nucleatum
BBE-Bilophila
CCFAClostridium
difficile
Identification disks:
kanamycin 1000 ug
vancomycin 5 ug
colistin 10 ug
P. intermedia
B. fragilis
3
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Spot indole test
p-DMACA reagent
Colonies can also
be rubbed
onto filter paper
moistened
with reagent.
Blue color is +
Rapid urease test (disks,
tablets)
preformed enzyme;
incubate
aerobically,
read 1-2h
neg pos
Grouping of
gram-negative rods
B.fragilis grp
Prevotella
Porphyromonas
Fusobacteria
Bilophila
Desulfovibrio
C. ureolyticus grp.
Bile Kana
R
R
S
R
S
R
V
S
R
S
V
S
V
S
Van
R
R
S
R
R
R
R
Col Cat Nit
R
+ V
- R
- S
- S
+ +
R
-+ +
S -+ +
4
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Bacteroides fragilis group
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bile resistant (BBE plate)
resistant to kana, vanco, colistin disks
most clinical isolates are catalase pos
comprise 1/3 of clinical isolates
most virulent (capsule)
most antibiotic resistant
Parabacteroides distasonis, merdae,
goldsteinii, johnsonii, gordonii
Prevotella species
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Isolated from oral and pelvic
infections, abdominal and soft tissue
Growth inhibited on BBE (but may
turn agar black from hydrolysis of
esculin if colonies plated directly)
Resistant to kana, vanco, variable
colistin
Catalase and indole usually negative
Fusobacterium spp
species
bile NO3
ind lipase esc cells
nucleatum
necrophorum
naviforme
gonidiaform.
mortiferum
varium
S
SR
S
S
R
R
+
+
+
+
+-
-
+
-+
r,pt
- r,pleo
boat
- gonidia
v pleo
- pleo
5
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Fusobacterium nucleatum
S to kana, colistin disks; R to
vanco
indole positive; lipase negative
slender rods with pointed ends
several different colony types;
subspecies
Isolated from all types of
infections and all areas of the
body
polymicrobial infections or
single isolate
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Fusobacterium necrophorum
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S to kana and colistin disks ; R
to vanco
indole and lipase positive; betahemolytic
cells have rounded ends or may
be very pleomorphic
important pathogen in
Lemmiere’s disease and
postanginal sepsis in young
people (deep vein thrombosis,
metastatic thrombi)
Throat cultures?
Bacteroides ureolyticus-like grp
Kana-S, Vanco-R, Col~S
B. ureolyticus
Camp. gracilis
Camp. rectus
Bilo. wadsworthia
Sut. wadsworthen.
Dial. pneumo. Col-R
Desulfovibrio Col-R
Eiken. corrod.
URE
+
+
-+
-
MOT
+
+/-
CAT
-+
+
-+
-
BILE
R
R
-R
-
CO2
+
NO2
+
+
+
+
+
+
+
( Use disk test for bile resistance)
6
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Clostridium perfringens
Box car shaped GPB
 Double zone of
beta-hemolysis on BA
 Lecithinase positive
 Produces abundant
gas in liquid media
(blood culture bottles?)

Clostridium innocuum
Resistant to cefoxitin, other cephs, vancomycin
(MIC= 8-16), some strains R to clindamycin,
quinolones
 Grows on CCFA medium – resembles C. difficile
but (PRO-neg)
 Disks- resistant to Ka, Va, Cl
 Misidentified in preformed enzyme
kits ( often as C. subterminale)
 Gram variable rod, occ large
terminal spores
 Isolated from IA infections, blood
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Clostridium ramosum
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Gram-variable long slender rod
Chains in broth media
Spores are rarely visible – terminal
May be resistant to antimicrobials
Identify using enzyme kits,
biochemicals
7
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Clostridium clostridioforme group
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Typically stains Gram-negative
Spores are rarely seen
Sensitive to vanco and kana disks
One of most frequently encountered clostridia
~10-30% produce beta-lactamase; generally
more resistant to antimicrobials (clinda,
moxifloxacin)
Identify with enzyme kits or biochems
New species: boltae, hathewayii, citroniae,
aldenense
Clostridium septicum
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Swarmer; subterminal spores; indole-neg
Bacteremia associated with cancer of large
bowel
Portal of entry – ileocecum
Myonecrosis, gas gangrene)
Underlying conditions- (leukemia,
lymphoma, diabetes
Susceptible to usual antimicrobials
Toxins not eliminated with abx treatment
High mortality
Gram-positive rods–
non-sporeforming
catalase
Propionibacteria +
Actinomyces
-+
Eggerthella lenta +
“Eubact. grp.”
Lactobacilli
Bifidobacteria
-
nitrate
++
+
-+
-
indole
+-
8
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Propionibacterium acnes
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Frequent skin contaminant in blood, csf cultures
Occasionally pathogenic (shunts, implants, postop cultures from eye)
Relatively slow-growing
ID based on pos rxns for
catalase, indole, nitrate
Acne strains may be R to
tetra and macrolides
All strains R metronidazole
Importance of basic tests – ID to
group >75% of clinical isolates
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Gram stain – cell morph
Growth in 20% bile
Susceptibility to 1mg kana
Susceptibility to 5 ug vanco
Catalase
Spot indole
Nitrate
Fluorescence
Limitations of phenotypic
identification tests
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Current and newer taxonomy is based
on 16S rRNA and other gene
sequences
Identification kits have limited
databases resulting in
misidentifications of some organisms
Expensive and time-consuming for the
results obtained
9
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
What can classical methods
still do?
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Provide a basic level of identification
based on phenotypic appearance
Provide isolates for further study
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Susceptibility to antimicrobials and to
detect novel resistance mechanisms
Sequencing to identify new species
Molecular characterization of
resistance mechanisms – limitations
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The cfiA gene is present in 3-5% of
bacteroides fragilis, yet resistance is not
expressed unless insertion sequences are
present
NimB genes may be present if Finegoldia
magna, yet the strains remain susceptible
to metronidazole
Multiple mechanisms may be present in one
strain – eg beta-lactamase + efflux pumps
Emergence of novel mechanisms
Diversity of Anaerobic Bacteremia:
Gram-negative isolates
Group, Genus 16S rRNA vs Conventional
Genus Species
Bacteroides (129) 129 124
Fusobacterium (15) 15 15
Parabacteroides (7) 7
7
Porphyromonas (1)
1
1
Prevotella (11)
11
11
Veillonella (4)
4
3
Other (4)*
4
3
*Alistipes, Bilophila, Campylobacter
Genus Species None
127 102
26
15
14
0
7
5
2
1
0
0
10
8
4
0
0
4
0
0
4
Simmon KE el al, J Clin Microbiol 2008,46:1596
10
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Diversity of Anaerobic Bacteremia:
Gram-positive isolates
Group, Genus
16S rRNA
Conventional
Genus Species
Genus Species None
Anaerococcus (7)
Clostridium (97)
Eggerthella (14)
Eubacterium (1)
Finegoldia (3)
Parvimonas (3)
Peptoniphilus (8)
Other (12)*
7
97
14
1
3
3
8
12
1
93
14
1
2
3
1
10
7
93
9
0
2
3
0
5
0
71
7
0
0
0
0
1
*Actinobaculum, Catabacter, Propionibacterium, Ruminococcus,
Solobacterium, Tissierella
Simmon KE el al, J Clin Microbiol,2008,46:1596
0
13
5
1
1
0
8
7
Identification of anaerobic GPR
from blood cultures
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20 isolates – compare 16S RNA gene sequencing to
conventional methods
11 clostridia – barati, difficile, indolis, innocuum,
paraputrificum, ramosum, septicum
9 NSF –Eubacterium, lactobacilli, P. acnes
Correct identifications:
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Vitek ANI – 2/20;
RapID ANA II – 7/20;
API 20A – 6/20;
MicroSeq 13/20
Lau SKP et al, J Clin Path 2006:59;219-222
16S rRNA sequencing vs
conventional for 127 blood
culture anaerobes (%)
Genus and
species
Genus only
No ID
Mis ID
16S MicroSeq
94
94
5
0
16S GeneBank
98
98
2 (new
species)
0
Conventional
52
79
9
9
Some of the unusual isolates: Actinomyces europaeus, A. funkeii, Clostridium
hathewayi ,C. scindens, Lactobacillus sakei, Robinsoniella sp, Solobacterium
moorei, Turicibacter sanguinis, Bacteroides dorei, B. nordii, B. xylanisolvens,
Sneathia sanguinegens, Veillonella dispar, Veillonella rodentium
Justesen, 2010, JCM
11
Citron - Classical methods for identification of anaerobes from mixed infections: do we still
need them?
Bacteroides and Parabacteroides recovered from clinical
specimens at St. John’s Med Ctr Santa Monica, CA 2006-11
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Species (N=559)
no.
% total
B. caccae
B. cellulosilyticus
P. distasonis
B. dorei
B. eggerthii
B. fragilis
P. goldsteinii
P. gordonii
B. intestinalis
P. johnsonii
B. massiliensis
P. merdae
B. nordii
B. ovatus
B. salyersiae
B. stercoris
B. thetaiotaomicron
B. uniformis
B. vulgatus
B. xylanisolvens
28
3
23
1
1
211
10
2
2
5
2
7
3
69
4
3
100
35
49
1
5.0
0.5
4.1
0.2
0.2
37.7
1.8
0.4
0.4
0.9
0.4
1.3
0.5
12.3
0.7
0.5
17.9
6.3
8.8
0.2
Anaerobic Gram-positive recovered from
clinical specimens cocci 2006-2011
Species
(N=240)
Finegoldia magna
Parvimonas micra
Pe. asaccharolyticus *
Pe. gorbachii
Pe. harei
Pe. harei-like
Ps. anaerobius
A. prevotii
No GenBank match
No.
96
67
23
4
3
9
12
4
6
%
40
27.9
9.6
1.7
1.3
3.8
5.0
1.7
2.5
One each: Anaerococcus hydrogenalis, A. lactolyticus, A. murdochii,
A. tetradius, A. vaginalis; Peptococcus niger;
Peptoniphilus coxii, P. tyrelliae ; Murdochiella assacharolytica; (0.4%)
Prevotella species recovered from clinical
specimens from 2006 - 2011
Species (n=143)
No.
% of total
P. bivia
46
32.2
P. buccae
21
14.7
P. buccalis
3
2.1
P. denticola
5
3.5
P. disiens
2
1.4
P. intermedia
8
5.6
P. loescheii
5
3.5
P. melaninogenica
18
12.6
P. nanceiensis
10
7
P. oralis
5
3.5
P. oris
3
2.1
P. salivae
2
1.4
P. timonensis
5
3.5
P. baroniae, P. bergensis, P. heparinolytica, P. tannearae
P. species, no GenBank match
1 ea
0.7
7
4.9
12
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Antibiotic resistance determination: dilution methods versus disc diffusion ‐ the old story comes back
Ulrik Stenz Justesen, MD, DMSc
Department of Clinical Microbiology
Odense University Hospital
ECCMID, London, 2012
Antimicrobial susceptibility testing (AST)
Antibiotic resistance determination or Antimicrobial susceptibility testing (AST) Why do we do AST?
“Antibiotic resistance among anaerobic organisms has increased significantly in recent years.” (2007)
“High levels of antimicrobial agent resistance among anaerobic organisms are continually reported.” (2012)
Foreword: CLSI Standard: Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard. M11‐A7 and A8.
Nguyen MH et al. Antimicrobial resistance and clinical outcome of Bacteroides bacteremia: findings of a multicenter prospective observational trial. Clin Infect Dis. 2000.
13
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Why we do AST?
Snydman et al. Clin Infect Dis. 2010.
Why do we do AST?
Why do we do AST?
14
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Why do we do AST?
How do we do AST?
What methods are available?
• Agar dilution or broth dilution (CLSI reference method)
• Gradient strip (Etest , MICE and others ...)
• Disk diffusion??? Agar or broth dilution
Brucella Blood Agar or broth
Clinical and Laboratory Standards Institute (CLSI)
•
Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard. M11‐A8. 2012.
Pros and cons
• Excellent reproducibility
• Laborious
15
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Agar dilution
Amyes S. OIDL Antibacterial Chemotherapy. 2011.
Broth dilution – commercial assays
Breakpoint testing ‐ not suitable for monitoring purposes
Gradient strip
Etest, MICE and others ...
The Etest is FDA approved but not addressed by the CLSI
McFarland 1.0 on supplemented (hemin and vitamin K) Brucella Blood Agar – BBA
Pros and cons
• Very easy
• Very expensive
Bacteroides fragilis ATCC 25285
16
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
When should we do AST?
“To assist in the management of infection in individual patients with serious or life‐threatening infections”
Relying on surveillance testing?
Snydman et al. Clin Infect Dis. 2010.
The old story comes back?
• Disk diffusion is coming back?
• High levels of antimicrobial agent resistance among anaerobic organisms are continually reported
• Clinical microbiology laboratories are asking for simple and inexpensive methods
• We have clinical EUCAST MIC breakpoints EUCAST MIC breakpoints (anaerobes)
17
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Is there a EUCAST disk diffusion method? No!
Is there a EUCAST disk diffusion method? Does anaerobes grow on the MH‐F (Mueller‐Hinton Fastidious)?
No!
Justesen et al. ECCMID. 2011.
Disk diffusion and anaerobic bacteria?
• Not a lot of data so far
• Sometimes used for screening, e.g. moxifloxacin and C. difficile
• Might work with some of the 24‐hour anaerobes, e.g. Bacteroides spp. and Clostridium spp.
• Some years ago it seemed to work for Bacteroides (Johnson et al. Clin Infect Dis. 1995) ‐ ”but it did not make it over the top”
Why? No resistance? Disagreement on conditions ‐
Wilkins‐Chalgren/BBA? Mix of slow and fast growing anaerobic bacteria?
Bacteroides fragilis ATCC 25285
18
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Brucella Blood Agar for disk diffusion AST?
##
Highest mean difference 2.7 mm
Highest range 5.5 mm
Justesen et al. ECCMID. 2011.
Further studies are needed with clinical isolates
##
Justesen et al. ECCMID. 2011.
Disk diffusion AST for the B. fragilis group?
This slide is intentionally left blank
19
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Brucella Blood Agar for disk diffusion AST?
The Brucella blood agar for disk diffusion antimicrobial susceptibility testing – reproducibility results for Clostridium difficile ATCC 700057
Abstract and poster P680
Justesen et al. ECCMID. 2012.
Results
Disk diffusion AST for Clostridium difficile
Disk diffusion antimicrobial susceptibility testing of Clostridium difficile
Abstract and poster P681
Erikstrup et al. ECCMID. 2012.
20
Justesen - Antibiotic resistance determination: dilution methods versus disc diffusion - the
old story comes back
Disk diffusion AST standardisation so far
Conditions
•
Plates have been reduced 18‐24 hours before use (or not?)
•
Inoculum preparation: 1 McFarland in thioglycolate bouillon (saline 0.85%?)
•
Inoculation and incubation: complying with the EUCAST 15‐15‐15 rule •
Anaerobic atmosphere (10 % H2, 10 % CO2, 80 % N2)
•
Temperature and time: 37 °C for 24 hours
•
Brucella Blood Agar‐plates with hemin, vitamin K1 and laked sheep blood
Summary, conclusions and perspectives
Preliminary disk diffusion studies with Bacteroides fragilis group reference strains and clinical isolates have been promising. Furthermore, studies with clinical isolates of Clostridium difficile shows that isolates with reduced susceptibility to metronidazole and vancomycin can be separated from wild‐
type isolates with disk diffusion. Although the reference methods are still the AST methods of choice for a large number of slow growing anaerobic species, disk diffusion seems to be a potential alternative for certain rapidly growing anaerobic bacteria. Clostridium perfringens or other Clostridium spp. may well be the next candidates to be evaluated for AST with disk diffusion (penicillin, clindamycin and metronidazole susceptibility). 21
Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Culture-independent Molecular Methods
to investigate Flora Changes
leading to mixed Anaerobic Infections
Georg Conrads & Hans-Peter Horz
Division of Oral Microbiology & Immunology,
University Hospital (RWTH), Aachen, Germany
ESCMID Educational Workshop, London, March 2012
The Dawning of Molecular Identification in Microbiology
16S rRNA
In 1977 Carl Woese did the first microbial phylogenetic tree based on
16S rRNA sequences, a „molecular clock“ of bacterial evolution
Contents
Molecular Methods discussed….
1)
DNA probes & primers
a)
b)
2)
(RTQ-) Polymerase Chain Reaction
a)
b)
c)
3)
4)
genomic: design, examples, problems
oligonucleotides: design, examples, problems
universal
specific
Fingerprinting
Microarrays, Chips & Surface Plasmon Resonance
Sequencing: pyro-, nano-, mixed
..with special attention applications & challenges
22
Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
1) DNA probes & primers
Genomic probes:
•
Genome fragments
•
Long PCR products or even the whole genome
•
(complete) sequence unkown
•
But empirically specific for species
Oligonucleotide probes (& primers):18-35 mer
in-silico designed & tested
•
Probes

Genomic

oligo
AATUGCTATCGCAATAGGCTAGCGGTACCGGATTACGG
ATACCAGTTATCGGGACCATTTATAGGACCATTTTAGGG
CAAAACTTTTTCGGGATTTCTCAAAGGGAGATTAGGAC
ACCACCATTATATTATTAGGGCCCATTTATTAGGAGGGG
GCTCCTTAAAAGGGGAAGGGGGAAATTTCTTTGGGATT
TCTTCTTCTCTTCTTUCTCTTCTGGAGAGGAGAGTTCGG
AGATTTAGGATTAGGCTTTAGGGGGACCCCCAAAAATT
CGGTATACATAGGACATTAGACCCAGTACCAAATTGCT
ATCGCAATAGGCTAGCGGTACCGGATTACGGATACCAG
TTATCGGGACCATTTATAGGACCATTTTAGGGCAAAAC
UTTTTCGGGATTTCTCAAAGGGAGATTAGGACACCACC
ATTATATTATTAGGGCCCATTTATTAGGAGGGGGCTCCT
TAAAAGGGGAAGGGGGAAATTTCTTTGGGATTTCTTCTT
CTCTTCTTTCTCTTCUGGAGAGGAGAGTTCGGAGATTTA
GGATTAGGCTTTAGGGGGACCCCCAAAAATTCGGTATA
CATAGGACATTAGACCCAGTACCA
AAGTTGACGTGGACTGGGATUUUUU
Genomic DNA probes
Selective strategy: Restriction fragments of the
test-strain hybridized to biotinylated DNA of
the closest related species and hybrids
eliminated by avidin-agarose gel: remaining
DNA fragments labelled and used as probes
(Schmidhuber 1988)
Random Prime Labelling: Gene Images RPL
Module (GE), Dig-High Prime (Roche)
Random Nick Labelling: Dig-Nick (Roche)
23
Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Genomic DNA probes: Examples
 Mainly
for Oral pathogens
in checkerboard format
 Food-borne (2011)
 Vaginal flora (2008)
 Mycoplasma, viruses
Limits will be discussed
Nascimento et al. 2006,
dental implant flora
Checkerboard DNA-DNA hybridization membrane showing the
hybridization of 40 of the 77 DNA probes to endodontic samples.
Brito L C N et al. J. Clin. Microbiol. 2007;45:3039-3049
Oligonucleotide probes and primers
 Probe
hybridisation
– Probe: oligonucleotide of 18-32 b length
– Target: rRNA/DNA signature, resistance gene, toxin-or other
virulence gene
– Assay: Dot-/Slot-/cell-/ in situ-/ colony-/ELOSA-hybrid.
 Polymerase
chain reaction
– Primer: oligonucleotide combination
– Target: see above
– Assay: Broad to „universal“ range PCR, Specific-PCR, PCRderived fingerprinting, microarrays
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Computerdesign
In-silico design of ideal oligonucleotide:
– Automated comparison of e.g. 16S rRNA sequences
(BLAST, ARB etc.)
– Choice of region with 2-3 central mismatches; in the
case of primers additional mismatch at 3’ end
– avoid GC-stretches
– avoid hairpin or duplex formation
– Melting temperature at least 60°C, as high as 72°C
(primers)
– Amplicon length: between 100-200 bp (50-60% GC
content)
Databases & Web Tools
Databases:
– Ribosomal Database Project: http://rdp.cme.msu.edu
– ARB, Munich, Germany: www.arb-home.de
– NCBI (Genbank): www.ncbi.nlm.nih.gov
– RIDOM: www.ridom-rdna.de (sorry most anaerobes
still ignored!)
– ISENTIO Ripseq www.ripseq.com
Web Tools:
– Check probe (RDP), Check chimera (RDP), secondary
structure, Design probe (ARB), 16S chromatograms
(single, multi).
Ribosomal V6 tags pyrosequencing of
coral reef microbes,
Galdos et al. 2011(Environm.Microbiology)
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Empirical Testing
Positive control: Reference strains including ATCC
– plus: 10-20 uncorrelated strains of test species of
different origin
Negative control: Reference strains including
ATCC of the nearest neighbor
– plus: 10-20 uncorrelated strains of the nearest neighbor
of different origin
Additional control: 20 representatives of closely or
more distantly related species sharing the same
habitat.
Oligonucleotide probes &
Blot-hybridisation
 Isolation of nucleic acids from
reference strains and clinical
material
 Immobilisation
 Hybridisation
 Detection (chemiluminescence,
fluorescence)
 Pros: cost-efficient & multiplex,
ratio of cell counts conserved
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Oligonucleotide probes: Challenges
 Each oligo needs individual hybridization
temperature to avoid cross-reactivity
Under stringent conditions: NO crossreactivity with other bacterial DNA but likely with HUMAN DNA
2) P C R - based diagnostic
Classic and quantitative (qPCR, RTQ-PCR)
ESCMID Educational Workshop, London, March 2012
The „universal“ PCR
16S rDNA
Conserved primer annealing site
Variable „informative“ core
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Broad-range („universal“) PCR…
…has a broad-range of applications
total cell-count determination
detecting new species in clinical
samples (review by Y. Song 2005)
& restriction
fingerprinting
of ecosystems
higher sensitivity than culture
accuracy of 84% (complete
sequencing = 100%, phenotypic=
56%)
Real time quantitative PCR
pre-and post
therapy
SybrGreen
106
3x104
TaqMan
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Principles, when using PCR for routine
three (to five) separted rooms: sample preparation,
adding of positive control, amplification
floor with sticky mats to avoid cross contaminations
closed systems (LightCycler, TaqMan)
NA-decontamination of surfaces (e.g. DNA Exitus)
amplicon-decontamination of sample by uracil-Nglykosylase (UNG) (plus using dUTP instead of dTTP
in PCR reaction)
negative control for PCR and nucleic acid preparation
inhibition control
Cell
count
3x105
3.33
Tannerella forsythia
5x104
0.56
Porphyromonas gingivalis
2x103
0.02
Treponema denticola
6x105
6.66
Fusobacterium nucleatum
3x102
<0.01
Parvimonas micros
4x104
Total
9x106
Aggregatibacter
actinomycetemcomitans
%
RTQ-PCR
periodontitis
typical result
(commercial product)
0.44
100
7,24%
Unimportant rest
0,45%
3,33%
A.actino
?
Red complex
88,98%
Orange complex
PCR based Fingerprinting
Method
rDNA based: ARDRA,
T-RFLP, DGGE, ITSPCR, rMLST (domain to strain)
genomic DNA based:
Rep-, Box-, ERIC-PCR;
AP-PCR, MLST
Pub-Med
History
T-RFLP
646
1997-2012
ARDRA
364
1992-2012
DGGE
2,148
1988-2012
ITS-PCR
1,300
1991-2012
(r)MLST
229
2001-2012
Rep-PCR (also ERIC, BOX)
696
1991-2012
AP-PCR (misleading RAPD)
442
1990-2012
RTQ-PCR, qPCR
4,983
1990-2012
Nested PCR
5,525
1990-2012
PCR
For contrast: PFGE
380,000
9,217
1986-2012
Before 1986 2012
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
T-RFLP = terminal restriction fragment length polymorphism
Universal PCR 16S rDNA
4 T-RFLP profiles
T-RFs
Virtual
Digest
Culman et al., 2009
BMC Bioinformatics
http://trex.biohpc.org/
Shyu et al., 2007;
Microb. Ecol.
http://mica.ibest.uidaho.edu/trflp.php
HOMD
Example: oral microbial changes during
a challenging trecking tour
Group I
Group II
n = 28
n = 30
Sampling at end of trek
Manang
No symptoms
Symptoms
Sampling at start
n = 58
Bhulebhule
58 healthy volunteers were
included in the study
Comparison of T-RFLP profiles using PCA
Start of trip
Group I
Group II
n = 28
PCA 2 (21%)
PCA 2 (31%)
n = 30
No symptoms
No symptoms
PCA 1 (37%)
PCA 1 (43%)
End of trip
n = 28
PCA 2 (18%)
PCA 2 (29%)
n = 30
No symptoms
Symptoms
PCA 1 (42%)
PCA 1 (43%)
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Microbial diversity significantly elevated in Group II
Group I
Group II
Shannon-Weaver Index
calculated as follows:
-∑ pi * (ln pi)
pi = proportion of individual
T-RFs
Start
End
Start
End
sp.
Bacteroidetes
T-RF 84 bp
T-RF 277nodatum
bp
T-RF
462 bp
Prevotella
sp.
Response
ofEubacterium
individual
T-RFs
Group I
Group II
Symptoms
No Symptoms
Group I
Group II
50 bp
Terminal Restriction Fragments (T-RF) in bp
Red signals: relative increase
500 bp
Green signals: relative decrease
3) Microarrays
(Gene chips)
All in one go!!
ESCMID Educational Workshop, London, March 2012
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Electrically addressable array
a)
b)
a) Nanoprobes, E-news 02-2006; b) Liu-Y et al. Analytical Chemistry 2008
Bead microarray (Illumina, iScan)
3 µm silica beads (50,000 per array)
with 1 Mio probes each, in wells linked
by optical fibres to detection unit
- man, mouse, rat arrays, no prokaryotes
Photolithographic high-density
array (Affymetrix)
Many eukarya: human, mouse, zebrafish, yeast, rice, Drosophila, C.
elegans, Aradiposis, barley, chicken, canine…..still only three
prokaryotes (not cost-efficient), Custom Array Service MyGeneChip
32
Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Spotted or printed microarrays
(NimbleGen-Roche, Agilent & IMGM)
very flexible; custom-made for 500 € (244 k) –
105 € (15 k) + costs for bioinformatics
….order by catalog (200 species) or
custom design (100-500 €/chip)
Surface Plasmon Resonance (SPR)
Mixed infection
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Gene chips in clinical microbiology
and infectious diseases
Comparative genomics
Strain typing and characterization
Host-pathogen interactions
Transcriptional regulation
Vaccine development
Pathogen detection and identification
Pathogen detection and identification
Dols et al. 2011: 14 Bacterial Vaginosis associated species
Harrington et al. 2008: 17 fecal flora analysis
Davignon et al. 2005: 20 respiratory tract pathogens
microarray incl. flu-like bioterrorism pathogens
Xing et al. & Institut Pasteur, 2006: 50 bacterial and viral
species plus antibiotic resistance and toxin genes.
Couzinet et al. 2005: 201 Staph. strains from 33 species and
different origin (human, vet., food, environment), 92%
concordance with 16S sequencing
DeSantis et al. 2005: simultaneous detection of 8,900 taxa
for microbial diversity (SSU1409-1491 directed, PM/MM).
Palka-Santini et al. BMC Microbiology 2009:
• 20 most prominent sepsis agents
• 930 gene segments including
virulence & resistance genes
•detection limit: 104 genomes by
LSpex pre-amplification with 800
primers in one reaction!
• 0.02 µM each primer
•Vent exo- DNA poly. (N.E. biolabs)
•annealing: 55°C 45s
•few genes not amplificated
(increase primer conc selectively)
•few cross-reactions
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Major Challenges & Solutions
 specimen collection
 instruction & information of
personnel
 filtration, concentration,
amplification (on-chip, whole
genome, LSplex)
 selective isolation (Loxter,
Molzym) of bacterial DNA
 RNA-dependent amplification,
blocking of dead cell PCR by
ethidium or propidium
monoazide intercalation &
crosslinking
 hierarchical sequences
 low pathogen number
 human competitor DNA
 only living cells
 so many species…
Bacteroides melaninogenicus
1970
subspecies
asaccharolyticus
B. asaccharolyticus
B. endodontalis
B. gingivalis
subspecies
melaninogenicus
B. melaninogenicus
B. denticola
B. intermedius
B. loescheii
B. corporis
P. disiens
Prevotella
P. enoeca
subspecies intermedius
1977
P. intermedia
Porphyromonas
P. asaccharolytica
P. endodontalis
P. gingivalis
P. gulae
P. circumdentaria
(P. salivosa)
P. canoris
P. somerae
P. albensis
P. baroniae
P. bivia
P. buccae
P. buccalis
P. brevis
P. bryantii
P. corporis
P. nigrescens
P. melaninigenica
P. denticola
P. dentalis
P. histicola
P. nanceiensis
P. paludivivens
P. stercorea
P. timonensis
P. veroralis
2009
P. cangingivalis
P. cansulci
P. catoniae
P. crevioricanis
P. gingivicanis
P. macacae
P. levii
P. uenonis
40 years of research: make 55 out of 1!
P. heparinolytica
P. marshii
P. multiformis
P. multisaccachrivorax
P. salivae
P. shahii
P. loescheii
P. tannerae
P. oralis
P. oris
P. oulorum
P. ruminicola
P. pallens
P. amnii
P. bergensis
P. copri
P. falsenii
P. maculosa
P. zoogleoformans
Lessons from Oral Microbiology:
Periodontitis as an „Ecological disaster“ in
Sulcus gingivae
Healthy
Actinomyces odontolyticus
Veillonella parvula
Actinomyces viscosus
Campylobacter concisius
Capnocytophaga gingivalis
Streptococcus
gordonii
Eikenella corrodens
S. mitis
Fusobacterium nucleatum,
Parvimonas
GCF &micra,
pH up
Socransky, 1998
Marsh, 2003
Prevotella intermedia,
Eh down
P. nigrescens,
Streptococcus
constellatus, Campylobacter
gracilis, C. rectus, Eubacterium
nodatum,
Tannerella forsythensis
Porphyromonas gingivalis
Treponema denticola
Aggressive
Aggregatibacter
actinomycetemcomitans
Periodontitis
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
How can we monitor mixed anaerobic bacterial
ecosystems by microarrays?
1. Sampling
Female, 36 y, loss of bone
horizon. + vertical., BOP,
inflammation
2. Transport to the laboratory
3. Isolation of DNA
4. PCR of 16S rDNA
Cy5
P.gingivalis
Sample of e.g.
subgingival
plaque
P.gingivalis
Species II
Species II
Species III
Species III
Species IV
Species IV
5. Hybridization
Porphyromonas gingivalis
catcher probe, position E8
6. Scan/Report
Report
Heading with data of patient, site,
clinical situation, laboratory
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Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Obstacles for accurate quantification
different lysis efficiency of gram-negative vers.
gram-positive and aggregated vers. single cells.
DeSantis: „Reproducible differences in microbial
community composition were observed by altering
the genomic DNA extraction method“
preferential amplification of different templates
(non-)proportional diagnostic probe hybridization
Conclusion: accurate quantification seems to be still
a “mission impossible” in chip formats
Polymicrobial infections: challenging
16S rDNA
Conserved primer site
information
Getting „automated“
 MicroSeq 500 (ABI): first sequencing kit for 527 bp of 16S rDNA
 Affirm™ VPIII (BD): the first direct specimen RNA probe-based
diagnostic test for the differential detection and identification of the
causative agents for vaginitis: Candida species, Gardnerella
vaginalis and Trichomonas vaginalis
 BD ProbeTec (BD, e.g. MAX): RTQ-PCR for Chlamydia
trachomatis (CT) and Neisseria gonorrhoeae
 SexTD-PCR-Kit (e.g. Bioneer): Detection of the most common
causes of genital infections: HPV , Herpes simplex Virus Type 1 and
2 , Chlamydia trachomatis , Neisseria gonorrhoea and Treponema
pallidum, Ureaplasma, Trichomonas etc.
PLEX-ID (Abbott Molecular): DNA mass spectrometry
37
Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
DNA analysis by Mass Spectronomy
(Plex-Id, Abbott)
universal PCR for
bacteria, viruses,
protozoa, fungi
PCR products are
analysed by mass
spectronomy, amplicon
mass profiles are checked
with Ibis -Biosciencedatabase
Strang 1b
Strang 1a
Ecker et al. 2008, Nature
4) Next-Generation Sequencing
New alternatives for Sanger method: pyro-, RDT, SBL
Voelkerdinger et al., 2009, Clinical Chemistry
454 FLX by Roche: Principles
DNA extraction
Adaptors for beads and primer
Pyro-Sequencing
One product per
nano well
38
Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
Ribosomal V6 tags
pyrosequencing of
coral reef microbes,
Galdos et al. 2011
(Environm.Microbiology)
Direct amplification & sequencing
in mixed Infections
homepage:
http://www.isentio.com/contentpages/Home.aspx
Two examples of mixed
chromatograms, without (A)
and with (B) displacement:
these can be solved by
uploading to INSENTIO: 2040 €/job, up to mix of three
very reliable, 16S only.
Kommedal et al. JCM, 46, 2008
39
Conrads - Culture-independent Molecular Methods to investigate Flora Changes leading to mixed Anaerobic
Infections
More Future: „Nanopore“-Sequencing
Fast & cost-efficient!
One genome per night?
DNA
Branton et al. 2008, Nature
Acknowledgements
Hans-Peter Horz, PD, Dr. rer.nat.
Ilse Seyfarth: technical support
ESCMID Educational Workshop, London, March 2012
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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Nagy - Why to use MALDI-TOF for the identification of anaerobes?
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