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Australian Society of Microbiology
Annual Conference 2008
CDS Workshop
Start
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ASM 2008 – CDS Workshop
• Reference Organisms – maintenance of (G. T. Fisher)
• ß-lactamase of Gram-negative bacilli (J. Pham)
• End
• Questions
• CDS website (G. T. Fisher)
• Opening (S. M. Bell)
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Tetracycline
• 30 μg disc replaced with a 10 μg disc
• Haemophilus influenzae with efflux
MIC = 32 mg/L = Resistant
30 μg disc annular radius = 5 to 7 mm
10 μg disc annular radius = 3 to 4 mm
• CDS Susceptible/Resistant cut-off point (10 μg disc)
Enterobacteriaceae
Vibrionaceae
Acinetobacter}
All other organisms 6 mm
4 mm
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Storage and Recovery of
Reference Organisms
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Reference Organism Reliability
• Minimise genetic change by
• Minimising subculturing
• Suspending or reducing metabolism
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-70°C Storage and Recovery of Reference Cultures
11
Store at -70°C
Monthly
After
1st Week
-70°C
storage
4°C
storage
n
n
n
n
Use for QA testing
After
3rd Week
ACM
strain
Culture
Fresh working culture
Discard
After
2nd Week
After
4th Week
2
3
4
5
Multiple
1 3 – 6
1
Discard
Discard
Discard
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After
1st Week
4°C
storage
Use for QA testing
After
2nd Week
Acquire
ACM
strain
Fresh working culture
Discard
Discard
Discard
After
30th Week
1
2
30
n
n
n
2 – 31
4°C Storage and Recovery of Reference Cultures
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Storage and Recovery of Neisseria gonorrhoeae
After 1st
Month
30-37°C storage
under paraffin
Acquire
ACM
strain
Fresh working culture
Discard
Discard
Discard
n
n
n
1
After 2nd
Month
After 6 Months
Use for QA testing
2 – 7
2
6
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CDS ATCC USP CLSI
Passages 6QA testing
5standard protocols
5
when referring to
ATCC number
2 to 8°C 1 week 1 week1 to 2
weeks
-20°C < 1 month Prolonged
-50 to -70°C 1 year
-70°C Long term 1 year
< -70°C Long term Indefinitely
-80 °C Long term Long term Indefinitely
Liquid Nitrogen
ImmersionLong term Indefinitely
Liquid Nitrogen
Vapour PhaseLong term Long term Indefinitely
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The ß-lactamases
of Gram-negative bacilli
An update
on detection of common plasmid
mediated β-lactamases in clinical
isolates in Australia
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Classification of β-lactamases
Ambler class Bush group(molecular structure) (inhibitor)
A (eg. TEM, ESBL) Group 2
(inhibited by CA)
B (MBL) Group 3
(not inhibited by CA
inhibited by EDTA)
C (AmpC) Group 1
(not inhibited by CA,
inhibited by boronic acid)
D (OXA) Group 4 (A. baumanii)
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Common transferable (plasmid
mediated) β-lactamases in coliforms
• TEM-1, ESBLs (Bush group 2, Ambler class A)
S/ AMC 60 → synergy (key hole)
Inhibited by CA
• AmpC: (Bush group 1, Ambler class C)
R/ AMC 60 S/ FEP 10
Not inhibited by CA, inhibited by boronic acid
• MBL: (Bush group 3, Ambler class B)
R/ AMC 60 R/ FEP 10
Not inhibited by CA, inhibited by EDTA
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E. coli ACM 5186 producing TEM-1 resistant to ampicillin (AMP 25)
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ESBLs(Ambler class A, Bush group 2)
Inhibited by CA
R/ Cephalosporins (including cefepime) and
aztreonam
S/ Augmentin (AMC 60)
Cephamycin (cefoxitin, cefotetan)
CDS routine testing → Synergy with AMC 60
(no need for confirmation)
S/ Imipenem (T)
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K. pneumoniae producing an ESBL
S/ Augmentin (AMC 60), typical synergy with cephalexin (CL 100) and
cefotaxime (CTX 5)
S/ imipenem (IMP 10) and cefotetan (CTT 30).
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CDS Routine Testing: Positioning strongly recommended
Klebsiella pneumoniae producing an ESBL: synergy between Augmentin (ACM
60) and cefepime (FEP 10), no obvious synergy with cefotaxime (CTX 5).
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Routine CDS test pattern of an organism of the ESCHAPPM group with an
inducible AmpC (flattened CTX 5 zone near IPM, 8 mm + resistant colonies)
R/ Augmentin (AMC 60), cephalexin (CL 100), S/ cefepime (FEP 10) and
imipenem (IPM 10).
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Plasmid mediated AmpC (PM-AmpC)
• R/ clavulanic acid• Constitutive (no induction, rare exception)• Similar profile as ESCHAPPM with minor variation
→ R/ 3rd generation cephalosporins, cephamycin (CMY)→ S/ cefepime (4th generation)
• CMY-1 (1989) in K. pneumoniae → E. coli• MIR-1 (1990) in K. pneumoniae → E. coli
90% identical to ampC gene in E. cloacae
• Coudron et al (1995-97) examined > 1000 isolates (VA)Found PM-AmpC in 1.6 % E. coli and 1.1% K. pneumoniae
• Confusing nomenclature:CMY → resistance to cephamycin (CMY-1…..CMY-21) DHA → Dharhan Hospital in Saudi ArabiaACT → Ambler Class C TypeFOX, MOX, LAT → resistance to cefoxitin, moxalactam, latamoxef(LAT-3 = CMY-6….)
• CMY-2 found in several serotypes of ceftriaxone resistant Salmonella
Loss of porin may add to resistance
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Detection of PM-AmpC in E. coli and
Klebsiella (non-ESCHAPPM)
R/ AMC 60 (not inhibited by CA)
R/ CL 100
R/ CTX 5 (high level resistance)
S/ FEP 10
Confirmation (optional): inhibition by boronic acid
1-Benzothiophene-2-boronic acid
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Routine CDS test showing an E. coli with plasmid mediated AmpC
R/ Augmentin (AMC 60), cephalexin (CL 100, cefotaxime (CTX 5);
S/ cefepime (FEP 10) and imipenem (IPM 10).
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E. coli with a plasmid mediated AmpC β-lactamase and boronic acid (BA)
Top half: CL 100, AMC 60 and CTX 5 control discs.
Bottom half: CL 100, AMC 60 and CTX 5 with 200 ug BA added
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E. coli with a plasmid mediated AmpC β-lactamase (low activity)
Top half: CL 100, AMC 60 and CTX 5 control discs.
Bottom half: CL 100, AMC 60 and CTX 5 with 200 ug BA added
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E. coli with a plasmid mediated AmpC β-lactamase and boronic acid (BA)
Top half: CL 100, AMC 60 and FOX 30 control discs.
Bottom half: CL 100, AMC 60 and FOX 30 with 200 ug BA added
CL 100 used in routine CDS testing is more sensitive to AmpC than FOX 30
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E. coli with a plasmid mediated AmpC β-lactamase.
CL 100, AMC 60 and CTX 5 discs placed near 200 ug BA discs
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Summary
Recognition of AmpC in routine testing
Criteria to recognise of AmpC in non-ESCHAPPM organism (eg.
E. coli, Klebsiella, Proteus mirabilis, Salmonella) in routine
testing:
CL 100 and ACM 60 < 6 mm
No “key hole” (no synergy with AMC 60)
→ AmpC
CTX 5 > 6mm = low level activity
CTX 5 < 6mm or no zone = high level activity
Record in comments section (suppressed)
The organism is usually susceptible to cefepime (FEP 10) unless
an ESBL or MBL is present.
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E. coli with a PM-AmpC and an ESBL in routine CDS test:
R/ AMC 60 and FEP 10 (and CTX, CL, AMP), synergy between AMC 60 and
cefepime (FEP 10). S/imipenem (IPM 10)
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The same E. coli with a PM-AmpC and ESBL
Top half: CTX 5, CAZ 10 and FEP 10 control discs.
Bottom half: CTX 5, CAZ 10 and FEP 10 with 2 ug clavulanic acid added
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E. coli with a plasmid mediated AmpC and ESBL
Top half: CTX 5, CAZ 10 and FEP 10 control discs.
Bottom half: CTX 5, CAZ 10 and FEP 10 with 2 ug clavulanic acid and 200 ug
boronic acid.
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Classification of beta-lactamases
Ambler class Bush group
(molecular structure) (inhibitor)
A (eg. TEM, ESBL) Group 2
(inhibited by CA)
B (MBL) Group 3
(inhibited by EDTA)
C (AmpC) Group 1
( NOT inhibited by
CA or EDTA)
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Acquired Metallo-Beta-Lactamases
(MBLs)Plasmid mediated MBLs
Ambler class B or Bush group 3
Inhibited by EDTA (Zinc molecule)
IMP-4 (most common)
VIM, SPM, GIM, SIM (P. aeruginosa)
Hydrolyses all beta-lactam (except aztreonam)
Enterobacteriaceae
May have a zone > 6mm with IPM 10
Pseudomonas aeruginosaHighly resistant to all β-lactams (S/ATM)
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An isolate of C. diversus R/ Augmentin (AMC 60), cephalexin (CL100),
cefotaxime (CTX 5) with colonies in cefepime (FEP 10) zone and some at the
edge of imipenem (IPM 10) zone (> 6 mm). ???
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R/AMC 60, CL100, CTX 5 and colonies in cefepime zone (FEP 10) and some at the edge of imipenem zone (> 6 mm).
• No synergy between FEP/AMC → not ESBL
• Numerous resistant colonies in FEP 10 → Candidate for MBL detection
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Simple detection of MBL:
Same isolate showing synergy between an EDTA disc placed next to cefotaxime
(CTX 5)/ imipenem (IPM 10)/ cefepime (FEP 10)/ Augmentin (AMC 60) discs..
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MBL producing E. cloacae:
Synergy between EDTA and cefotaxime (CTX 5)/ ceftazidime (CAZ 10)/
cefepime (FEP 10) / imipenem (IPM 10). S/ aztreonam (ATM 30)
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MBL and ESBL: K. pneumoniae showing borderline imipenem zone (IPM 10) and
a large zone with IPM 10 loaded with EDTA, synergy between a central EDTA disc
and cefotetan (CTT 30). The presence of an ESBL is revealed by the resistance to
aztreonam (ATM 30) and synergy between ATM 30 and AMC 60.
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Pseudomonas aeruginosa candidate for MBL detection: highly resistant to all
β-lactams, imipenem (IPM 10) ceftazidime (CAZ 10), tazocin (TZP 55), cefepime
(FEP 10) and Timentin (TIM 85) except aztreonam.
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Detection of MBL: Synergy between an EDTA disc placed next to imipenem
(IPM 10)/ meropenem (MEM 5)/ ceftazidime (CAZ 10) discs.
S/ aztreonam (ATM 30)
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