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GRAM NEGATIVE ORGANISMS:
KEY AST CHALLENGES
Amanda T. Harrington, PhD, D(ABMM)
Associate Professor, Pathology
Director, Clinical Microbiology Laboratory
Loyola University Medical Center
Discuss current challenges regarding
antimicrobial susceptibility testing for Gram
negative organisms
Discuss basic limitations to assessing
carbapenem resistance in GNRs
Discuss antimicrobial susceptibility testing
and reporting strategies for GNRs
OBJECTIVES
ENTEROBACTERIACEAE
Cefazolin testing to predict oral cephalosporins for
uUTI
ESBLs and AmpCs
Carbapenem Resistance
THE CHALLENGES
Cefazolin:
Surrogate Agent to Predict Oral Cephs
Infectious Diseases Society of America Practice Guideline
re: β-lactams for Acute uUTI in Women
http://www.idsociety.org
β-lactams including
amox-clav,
cefdinir,
cefaclor,
cefpodoxime
are choices when other agents (e.g.,
nitrofurantoin, trimeth-sulfa, fosfomycin,
fluoroquinolone), cannot be used
Other β-lactams (e.g., cephalexin) are
less well studied but may also be
appropriate in certain settings.
uUTI = uncomplicated urinary tract infection
CEFAZOLIN % SUSCEPTIBLE
WITH 2010 AND PRE-2010 BREAKPOINTS
54
69
11
72
81 83
0
20
40
60
80
100
E. coli (n=581) K. pneumoniae
(n=313)
P. mirabilis
(n=169)
% S
usce
pti
ble
"New" Cefazolin <=2
"Old" Cefazolin <=8
M100 -S25 TABLE 1 A
Test/ Report
Group Agent
MIC Breakpoint (µg/ml) Comments
Susc Int Res
Cephems (Parenteral)
A Cefazolin ≤2 4 ≥8 based on dose of 2 g every 8 h
Cephems (Oral)
U Cefazolin* ≤16 - ≥32 Footnote (20)
ENTEROBACTERIACEAE CEFAZOLIN
* “surrogate” agent
CLSI M100-S25 Table 2A
• (20) Cefazolin predicts results for the oral agents - cefaclor, cefdinir,
cefpodoxime, cefprozil, cefuroxime axetil, cephalexin, and loracarbef when
used for therapy of uncomplicated UTIs due to E. coli, K. pneumoniae, and
P. mirabilis.
PRESCRIBING CEFAZOLIN VS. MIC
INTERPRETATION
IM, IV administration
Introduced 1973; today, mostly used for:
MSSA
Prophylaxis for some surgical procedures
If GNR known “S” (only E. coli, K. pneumoniae, P. mirabilis): Uncomplicated UTIs; bacteremia
M100-S25. Table 2A.
Reason for Testing Breakpoints (µg/ml)
Dose S I R
Predict cefazolin use for uUTI ≤16 - ≥32 1 g every 12 h
(IM or IV)
Predict cefazolin use for systemic
infections ≤2 4 ≥8
2 g every 8 h (IM or IV)
Surrogate for oral cephalosporins to
use for uUTI ≤16 - ≥32 PO (various)
ampicillin >32 R
cefazolin 8
ciprofloxacin >2 R
nitrofurantoin ≤16 S
trimeth-sulfa >4/76 R
SPECIMEN: URINE
DIAGNOSIS: CYSTITIS
E. COLI
MIC (µg/ml)
How should cefazolin MIC of 8 µg/ml be
interpreted and reported for treatment
of UTI?
ampicillin >32 R
cefazolin 8 S
ciprofloxacin >2 R
nitrofurantoin ≤16 S
trimeth-sulfa >4/76 R
SPECIMEN: URINE
DIAGNOSIS: CYSTITIS
E. COLI
MIC (µg/ml)
“Cefazolin results should only be used to predict potential
effectiveness of oral cephalosporins (eg, cephalexin) for
treating uncomplicated urinary tract infections.”
Final Report with
Optional Comment
Example 1
ampicillin >32 R
oral cephalosporins S
ciprofloxacin >2 R
nitrofurantoin ≤16 S
trimeth-sulfa >4/76 R
MIC (µg/ml)
“Oral cephalosporins include cephalexin,
cefpodoxime, cefdinir.”
Final Report with
Optional Comment
Example 2
SPECIMEN: URINE
DIAGNOSIS: CYSTITIS
E. COLI
ampicillin >32 R
cefazolin 8 R
ceftriaxone ≤0.5 S
ciprofloxacin >2 R
gentamicin 1 S
piper-tazobactam ≤ 8 S
trimeth-sulfa >4/76 R
SPECIMEN: BLOOD
DIAGNOSIS: PYELONEPHRITIS
E. COLI
MIC (µg/ml)
“Cefazolin susceptible results are based on a dose of
2 g every 8 h.”
Final Report with
Optional Comment
Example 3
Commercial Systems
Most not FDA cleared for new breakpoints (BPs)
Some panels do not have low dilutions for
systemic breakpoints
Verify new breakpoints? urine? systemic?
Determine when testing for systemic isolates
needed
LIS
Does your system have flexibility?
CEFAZOLIN – CHALLENGES
Test urine isolates with commercial system
(ex. Vitek 2)
Test other isolates (blood) with broth
microdilution (low dilution wells)
E. coli, Klebsiella spp. and Proteus mirabilis
Report “oral cephalosporins” on urine isolates
Report “cefazolin” on special request
CEFAZOLIN – ONE POTENTIAL SOLUTION
ESBLs and AmpC
β-lactamases -
Enterobacteriaceae
AmpC and ESBL
ESBL (plasmid)
E. coli Klebsiella P. mirabilis
Other organisms
AmpC (chromosome)
Induction of AmpC β-lactamase
Selection of stably derepressed AmpC-R mutants
AmpC transfer to plasmid
No CLSI
endorsed test for
AmpC!
CLSI ESBL test
for E, K, P…
Agent Old Breakpoints Current Breakpoints
Susc Int Res Susc Int Res
Cefazolin ≤8 16 ≥32 ≤2 4 ≥8
Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4
Ceftizoxime ≤8 16-32 ≥64 ≤1 2 ≥4
Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4
Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16
Cefepime ≤8 16 ≥32 ≤2 4 ≥8
Aztreonam ≤8 16 ≥32 ≤4 8 ≥16
ENTEROBACTERIACEAE - CEPHALOSPORINS
CLSI BREAKPOINTS (MIC µG/ML)1
M100-S25 Table 2A.
1 CLSI also revised corresponding disk diffusion breakpoints
REVISED CEPHALOSPORIN /
AZTREONAM BREAKPOINTS
If you are using the “old” breakpoints (pre -2010) you
must continue to test for ESBLs
Minimal impact on overall %S for each agent
Considerable reluctance to eliminate ESBL testing
Infection prevention groups still want the information
Still not much data for “ESBL” isolates treated with
susceptible cephalosporin
More controversial than carbapenem
breakpoint changes!
AmpC
Hydrolyze ceftriaxone, cefotaxime,
ceftazidime, cephamycins (cefoxitin) NOT
cefepime
Not inhibited by -lactamase inhibitors
Chromosomal in SPICE or MYSPACE species
“S” isolates can become R during therapy
E. coli, Klebsiella, P. mirabilis and Salmonella
can acquire ampC on plasmid
AMPC -LACTAMASES
“SPICE” BUGS*
Serratia marcescens
Pseudomonas aeruginosa
Indole positive Proteeae –
Morganella morganii & Providencia spp.
Citrobacter freundii
Enterobacter spp.
Concern – de-repression of AmpC = resistance develops on therapy
*Also Hafnia alvei, Aeromonas spp., Yersinia, Acinetobacter baumannii and
certain non-fermenting gram-negative rods
Testing vs. no testing for AmpC or ESBL
Use current breakpoints vs. old
breakpoints
SEVERAL APPROACHES
ESBL IN MYSPACE BUG
PLASMID MEDIATED AMPC IN E.COLI
Selective Reporting:
ceftriaxone for E. coli / Klebsiella / P. mirabilis
cefepime for SPICE organisms (poor inducer of AmpC)
IF doctor calls, report ceftriaxone with disclaimer
“Due to the high likelihood of selecting resistant mutants,
ceftriaxone for serious infections caused by Enterobacter
spp. may result in high level resistance”
Additional Comments based on testing:
Inducible beta-lactamase
Plasmid mediated beta-lactamase
SEVERAL APPROACHES
Carbapenemase Detection
28
“WE HAVE A WEIRD-LOOKING
KLEBSIELLA PNEUMONIAE ON BLOODS”
29
BMD AST RESULTS: KLEBSIELLA
PNEUMONIAE
Agent
Amikacin 32 I
Aztreonam, cefepime, cetazidime, ceftriaxone
>32 R
Ciprofloxacin >2 R
Ertapenem >16 R
Gentamicin, tobramycin >10 R
Imipenem >16 R
Meropenem >16 R
Minocycline >32 R
Piperacillin-tazobactam >128 R
Trimeth-sulfa >4/76 R
Tigecycline 2 S
Colistin >16 ?
Isolate MHT+
“This isolate has unusual
carbapenem resistance results.
Infectious Diseases consultation
strongly suggested”
WHAT IS A CRE?
- Any Enterobacteriaceae or just Klebsiella
pneumoniae?
- Resistant to any carbapenem, all carbapenems,
select carbapenems?
- Resistant vs. not-susceptible?
- Must have a carbapenemase?
30
WHAT IS A CRE?
Current CDC Surveillance Definition
Enterobacteriaceae that are:
Resistant to one or more of the
following: doripenem, ertapenem,
meropenem, or imipenem* OR
Production of a carbapenemase detected
by PCR. MHT, Carba-NP, metallo-beta-
lactamase test
31 *Proteus/Providencia/Morganella exceptions for imipenem
Clinically important Often resistant to multiple classes
of antibiotics
Pan-resistant CRE have been described
Associated with high mortality rates (up to 70%) May be > 50% in ICU patients
Combination therapy appears to improve outcomes
Epidemiologically important Highly transmissible
Have spread throughout healthcare settings across the United States, (endemic in some areas)
Potential for CRE to become widespread if not contained
32
WHY ARE CRE IMPORTANT?
PR
AK
HI
States with CRE confirmed by CDC, 2013
Mechanism testing is not recommended for guiding therapeutic decisions
Not routinely performed in many U.S. clinical laboratories
However, carbapenemase-producing CRE (CP-CRE) are currently believed to be primarily responsible for the increasing spread of CRE in the United States
Phenotypic definitions based bacteria’s antibiotic susceptibility pattern primary way clinical laboratories and infection prevention teams attempt to identify CRE
No phenotypic definition will perform perfectly in distinguishing carbepenemase producers
Will l ikley increase measured CRE prevalence due to the fact that a larger number of non-CP-CRE will meet the current definition
CHALLENGES
Between carbapenem-resistant Enterobacteriaceae (CRE) and carbapenemase-producing (CP)-CRE?
The production of carbapenemases (called CP-CRE), enzymes that break down carbapenems and related antimicrobials making them ineffective.
This includes enzymes like Klebsiella pneumoniae carbapenemase (KPC)
The combination of mechanisms other than carbapenemase production (called non-CP-CRE), most commonly the production of beta-lactamases (e.g., AmpC) in combination with alterations in the bacteria’s cell membrane (e.g. , porin mutations).
WHAT IS THE DIFFERENCE…
35
BMD AST RESULTS: ENTEROBACTER
CLOACAE
Agent
Amikacin 16 S
Aztreonam, cefepime, cetazidime, ceftriaxone
>32 R
Ciprofloxacin >2 R
Ertapenem 0.5 S
Gentamicin, tobramycin 4 S
Imipenem 1 S
Meropenem >16 R
Minocycline >32 R
Piperacillin-tazobactam >128 R
Trimeth-sulfa >4/76 R
Tigecycline
Colistin
Isolate MHT+ ?
REVIEW: MECHANISMS OF BETA-LACTAM
RESISTANCE IN ENTEROBACTERIACEAE
Remember! Not all CRE
have a “carbapenemase”
CARBAPENEMASES Class Examples Produced by: Notes
A
ESBLs [TEM, SHV, CTX-M] KPC carbapenemases
SME carbapenemases
K. pneumoniae and other Enterobacteriaceae
S. marcescens
Most inhibited by clavulanic acid Usually plasmid-mediated (not SME)
B
Metallo-β-lactamases (MBL) (e.g. NDM, VIM, IMP, GIM, SPM carbapenemases)
P. aeruginosa
Enterobacteriaceae
Acinetobacter
S. maltophilia
Inhibited by EDTA Do not hydrolyze aztreonam
C AmpC Enterobacteriaceae Some non-fermenters
Inducible in some genera Not inhibited by clavulanic acid
D
OXA carbapenemases (e.g. OXA-48, -181, -232)
Acinetobacter baumannii
Enterobacteriaceae
Hydrolyze carbapenems to some degree
Adapted from Queenan & Bush. 2007. Clin Microbiol Rev. 20:440.
Bush & Jacoby. 2010. AAC. 54:969; Bush, K. 2013. Ann NY Acad Sci 1277:84. Slide from Janet Hindler
MHT Carba NP Molecular
Use Enterobacteriaceae Enterobacteriaceae
P. aeruginosa
Acinetobacter
Enterobacteriaceae
P. aeruginosa
Acinetobacter
Strengths Simple Rapid Determines type of
carbapenemase
Limitations Some false pos (eg,
ESBL/ampC + porin)
Some false neg
(eg NDM)
Enterobacteriaceae
only
Special “fresh”
reagents
Some invalid results
False neg for OXA-
type carbapenemase
Special reagents
Specific to targeted
gene
Introduction to Tables 3B and 3C. Tests for
Carbapenemases in Enterobacteriaceae, Pseudomonas
aeruginosa, and Acinetobacter spp.
CLSI M100-S27. --now includes mCIM; will discuss later today
NO
Still MDR
Still risk for infection control
YES
Hospitals may have different practices for non-CP CRE
New antimicrobial agents designed to target certain mechanisms
Mixing CP CRE and non-CP CRE ‘confuses’ the epidemiological
landscape
IS DETECTION OF CRE THAT DO/DON’T
PRODUCE CARBAPENEMASE
IMPORTANT?
DO LABORATORIES NEED TO DO
CARBAPENEMASE MECHANISM TESTING?
Cefazolin can be used as a surrogate agent to predict the activity of the oral cephalosporins for the treatment of uUTI caused by E. coli, Klebsiella, P. mirabilis
ESBL testing is no longer required if current cephalosporin / aztreonam breakpoints are used
Strategic reporting of ESBLs and AmpCs may be beneficial to individual hospital systems; consult with colleagues before changing practice
CRE is challenging, but there are a variety of practices available to help optimize patient care
SUMMARY
NONFERMENTING GRAM
NEGATIVE RODS
P. aeruginosa and Acinetobacter baumannii are
common nosocomial pathogens
Can be extremely drug-resistant
Same mechanisms responsible for carbapenem
resistance in Enterobacteriaceae may be present in
nonfermenting GNRs (e.g. plasmid beta-lactamase)
however resistance is typically combinatorial
Quick to become multidrug-resistant given arsenal of
intrinsic resistance mechanisms
BACKGROUND
Main contributors:
Chromosomal AmpC
Loss of porin OprD
Hyperexpression of efflux pump MexAB-OprM
In a recent study of isolates with reduced to no
susceptibility to ceftazidime, a total of 21
different combinations of resistance mechanisms
were found
Chromosomal and acquired beta-lactamases
Metallo-beta-lactamase most common
carbapenemase
BETA-LACTAM RESISTANCE IN
P. AERUGINOSA
Castanheira M et. al., AAC, 2014
Main contributors:
Chromosomal AmpC-type beta-lactamase
Oxacillinases
Metallo-beta-lactamases
Carbapenem resistance is most often linked
to a carbapenemase
Carbapenemases (e.g. OXA) pose additional
threat because many are located on mobile
genetic elements
BETA-LACTAM RESISTANCE IN
ACINETOBACTER
Potron A et. al., Int J Antimicrob Agents, 2015
MAIN MECHANISMS OF CARBAPENEM
RESISTANCE
Enterobacteriaceae Cephalosporinase + porin loss +/- ESBL
Carbapenemase
P. aeruginosa Porin loss
Up-regulated efflux
Carbapenemase
Acinetobacter spp. Cephalosporinase + porin loss
Carbapenemase
Accuracy of automated systems for detection of
carbapenem resistance is varied
For Acinetobacter baumannii/calcoaceticus
complex, reported very major error rates against
imipenem
Vitek2 – 0.7-4% VME
MicroScan – 2.8-25% VME
Phoenix - 1.9% VME
Disk diffusion and Etest are typically reliable
when compared against broth microdilution
DIFFICULTIES WITH PHENOTYPIC TESTING FOR
DETECTION OF CARBAPENEM RESISTANCE
Markelz AE et. al., AAC, 2011
Disk diffusion and Etest may
be difficult to interpret (fuzzy
zones or inner colonies)
P. aeruginosa may be
mucoid hindering inoculum
density measurement and
zone definition
DIFFICULTIES WITH PHENOTYPIC TESTING
Traditional phenotypic tests (e.g. MHT)
demonstrate poor performance for detection
of carbapenemases in P. aeruginosa and A.
baumannii
E-test for MBL can be ‘false positive’
Is determination of carbapenem resistance
mechanisms in nonfermenting GNRs
important?
TESTS FOR CARBAPENEMASES
Is determination of carbapenem resistance
mechanisms in nonfermenting GNRs be important?
Infection prevention and control
Many mechanisms are chromosomal (and/or
combinatorial)
Would not want MDR Acinetobacter to become
endemic even if no plasmid-mediated resistance
mechanisms absent
Epidemiology – better tracking of transmission
events
Unlikely to affect treatment decisions
TESTS FOR CARBAPENEMASES
Prior to initiating a carbapenemase
testing protocol (especially for
nonfermenting GNRs), determine how
(and if) mechanistic information will be
used by infection control and prevention
and pharmacy
M100-S26 (TABLE 3)
Results in a large number
of indeterminate and
falsely negative results
Not recommended by
CLSI for organisms other
than Enterobacteriaceae
Performance is less than
desirable for MBLs, which
may be present in
Pseudomonas >
Acinetobacter
MODIFIED HODGE TEST
+
-
?
Based on in vitro hydrolysis of imipenem by a
bacterial lysate
Endorsed by CLSI for Enterobacteriaceae, P.
aeruginosa , and Acinetobacter spp.
High level of sensitivity and specificity (>90% for
both) in early reports, reevaluation indicates my be
less for sensitive depending upon user
Poor sensitivity for OXA-48-type carbapenemases
(can adjust method), GES in P. aeruginosa
Labor intensive (requires imipenem reagent
preparation at time of use)
CARBA NP
CARBA-NP: COMMERCIAL VERSIONS
Rosco Rapid CARB Screen
Biomerieux RAPIDEC CARBA NP
A B
CLSI M100-S25 Method
• Few studies of commercial
assay performance
• CARBA NP method
outperforms CARB Screen
Several tests utilize inhibitors that allow for
detection and differentiation of carbapenemases
DOUBLE DISK/INDIRECT/COMBINED/
INHIBITION TESTS
1 Mathers AJ et. al. , JCM 2013
1
Imipenem disk compared to
imipenem + EDTA disk (commonly
look for ≥5 mm difference in zone of
inhibition with addition of EDTA)
Inhibition by EDTA is a characteristic
used to distinguish MBLs from other
beta-lactamases
Carbapenemases other than MBLs
may be responsible for carbapenem
resistance – this is not a standalone
test
COMBINED DISK TEST
Imipenem
Imipenem +
EDTA
MBLs
Take home message: no phenotypic test can
be used as a standalone test for detection and
differentiation of carbapenemases in
nonfermenting GNRs
So what about molecular tests…
PHENOTYPIC TESTS
As with all molecular methods, dependent upon
knowledge about target sequence
Carbapenem resistance may result from
mechanisms other than carbapenemases
Would correctly indicate no carbapenemases
present
Stewardship: could use result and initiate use of
carbapenem while awaiting susceptibility results
Knowledge of local mechanisms are beneficial
MOLECULAR METHODS
Oxacillinase genes in Acinetobacter may be detected but may not result in phenotypic resistance
Probably one of the best methods for detection of oxacillinases in Acinetobacter
Will not detect all OXA types
Commercially -available assays are a great method for detection of some carbapenemases (NDM, VIM, IMP, OXA, KPC) but many others would be missed
P. aeruginosa may be challenging
MOLECULAR METHODS
Emergence of resistance in P. aeruginosa against
imipenem is common, especially with prolonged
treatment
0.3% of the genome is devoted to antimicrobial resistance
genes
10% genes organized in pathogenicity islands that can be
easily mobilized
Genes may be up or downregulated based on antibiotic
pressure
Differing opinions on frequency of susceptibility
testing for GNRs (range 1 – 3 days)
FREQUENCY OF SUSCEPTIBILITY TESTING
Mesaros N et. al., Clin Microbiol Infect, 2006
Plug in intrinsic resistance first
Check for updates
REPORTING STRATEGY
P. AERUGINOSA
aztreonam >32 R
ceftazidime <8 S
imipenem ≤2 S
ciprofloxacin >4 R
gentamicin >16 R
pip-tazo ≤ 8 S
ertapenem R
Consider reporting
some intrinsically
resistant agents
(e.g. ertapenem)
ACINETOBACTER
amp/sulbact <8S
ceftazidime <8 S
imipenem ≤2 S
ciprofloxacin >4 R
gentamicin >16 R
pip-tazo ≤ 16 S
ertapenem R
Consider reporting
some intrinsically
resistant agents
(e.g. ertapenem)
*sulbactam is the
active component
*
Current commercial panels should have appropriate concentrations to differentiate susceptibility and resistance
Detection of mechanism(s) responsible for carbapenem resistance is not necessary for treatment but may aid in identification of resident resistance mechanisms
REPORTING STRATEGY
No standard definition for reporting of
carbapenem resistance in nonfermenting
GNRs (e.g. carbapenem resistant organism,
multi-drug resistant organism)
Many public health laboratories focus on CRE,
so guidance is somewhat lacking
Develop protocol with infection control for
notification of carbapenem-resistant
organisms
REPORTING CARBAPENEM RESISTANCE
IN NON-ENTEROBACTERIACEAE
Accurate detection of carbepenemases in non-
Enterobacteriacea is challenging with currently available
methods
Detection of carbepenemases may not be clinically relevant to
providers
Proceed with caution
Strategic reporting and frequency of testing may be effective
strategies for antimicrobial stewardship
SUMMARY
Recommended