Detecting Resistance in GramNegative Bacteria, Including ESBL Issues

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Detecting Resistance in Gram-Negative Bacteria,
Including ESBL Issues!

• Mohammad Rahbar Reference Laboratories of Iran

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Extended-Spectrum-ß Lactamase(ESBLs)
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Evolution of ?-Lactamases
Plasmid-mediated TEM and SHV ?-lactamases
Extended-spectrum Cephalosporins
Ampicillin
1965
1970s
1983
1988
2000
TEM-1 E.coli S.paratyphi
TEM-1 Reported in 28 Gm(-) sp
ESBL in Europe
ESBL in USA
gt 130 ESBLs Worldwide
1963
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?-Lactamases

• TEM-1 ?-lactamase (coded by blaTEM-1 gene)
• amp-R in E. coli
• amp-R in H. influenzae
• pen-R in GC
• TEM-2 ?-lactamase (coded by blaTEM-2 gene)

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(cont,n)

• SHV-1 ?-lactamase (coded by blaSHV-1 gene)
• amp-R in K. pneumoniae
• blaTEM-1 and blaSHV-1 mutate and resultant genes
  code for ESBLs

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Other ESBLs

• CTX-M preferentially hydrolyze cefotaxime.
• - found in strains of Salmonella enterrica
• - E.coli and other species of Enterobacteriaceae
• OXA
• Mainly in P.aeroginosa

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Other ESBls (cont,n)

• PER-1 Discovered in P.aeruginosa (turkey)
• PER-2 In south America
• VEB-1 E.coli (Vietnam)
• CME-1 Chrysobacterium meningosepticum(
  Thailand)
• TLA-1 E.coli ( Mexico )

Patrica A Cli Microbiol Rev 14 2001
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Definitions

• Narrow-spectrum ?-lactam agents
• Active against G- or G bacteria only e.g.,
  penicillin
• Broad-spectrum ?-lactam agents
• Active against G- and G bacteria e.g.,
  ampicillin, 1st generation cephalosporins

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Definitions (cont)

• Extended-spectrum ß-lactam agents
• Enhanced activity against G- and some G
  bacteria e.g., 3rd and 4th generation
  cephalosporins, carboxy- and ureidopenicillins
• Extended-spectrum ß-lactamases (ESBLs)
• Enzymes produced by GNR that destroy certain
  extended-spectrum ß-lactam agents, including 3rd
  generation cephalosporins

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Enterobacteriaceae

• Extended-spectrum ?-lactamases (ESBLs)
• AmpC ?-lactamases
• Salmonella/Shigella

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Enterobacteriaceae First Choice Therapy

• 3rd gen cephalosporin
• fluoroquinolone
• ertapenem, imipenem
• meropenem
• ertapenem, imipenem
• meropenem
• extended-spectrum penicillin aminoglycoside

• Klebsiella
• Enterobacter

Sanford, 2004
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ESBls are clinically important

• They destroy cephalosporins
• Delayed recognition and inappropriate treatment
  of sever infection
• ESBls mediated resistance is not always obvious
  to all cephalosporins
• Many ESBL produce multi-resistant to non-
  ?-lactam antibiotics such as quinolons
  .aminoglycosides and trimethoprime

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Why is important to do special tests and follow
complex reporting rules for ESBLs?

• In vitro tests may be misleading (e.g., may see
  S in vitro but drug may not work!)
• Many types of ESBLs with varying susceptibility
  profiles (TEM ? 130 SHV ? 50 CTX-M ? 30)
• Treatment failures (esp. bacteremia) when patient
  treated with 3rd generation cephalosporins
• ESBL-producing isolates can cause nosocomial
  outbreaks

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Outcome of Serious ESBL Infections When Treated
with 3rd gen Cephalosporin
Paterson et.al. 2001. JCM.392206-12
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Which microorganisms are ESBls positive

• K.pneumoniae and K.oxytoca
• E.coli
• Enterobacter spp
• Salmonella spp
• Morganell morganii,Proteus mirabilis
• Serratia marcescens
• Pseudomonas aeruginosa

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ESBL Testing

• Organisms E. coli, Klebsiella
  spp., Proteus mirabilis
• Screen test Decreased susceptibility to
  extended-spectrum ß-lactams
• Phenotypic confirmatory test ß-lactam
  activity restored by ß-lactamase inhibitor (i.e.
  clavulanic acid)

CLSI M100-S15 Table 2A (M2, M7)
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Proteus mirabilis and ESBLs

• Not necessary to test routinely
• Uncommon in USA
• Significant problems in some locations (e.g.,
  Europe, South America)

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ESBL Reporting Rule

• The rule (CLSI NCCLS) M100-S15)
• Strains of Klebsiella spp. E. coli, and Proteus
  mirabilis that produce ESBLs may be clinically
  resistant to therapy with penicillins,
  cephalosporins, or aztreonam, despite apparent in
  vitro susceptibility to some of these agents.
• The message
• Report confirmed ESBL-producing strains as R to
  all penicillins, cephalosporins, and aztreonam

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ß-lactams Edited to R

• Penicillins
• e.g., ampicillin, carbenicillin, mezlocillin,
  piperacillin, ticarcillin
• Cephalosporins
• e.g., cephalothin, cefamandole, cefuroxime,
  cefotaxime, ceftriaxone, ceftazidime, cefepime
• Monobactam
• aztreonam

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ß-lactams NOT Edited to R re CLSI Reporting
Rules

• Cephamycins
• e.g., cefoxitin, cefotetan, cefmetazole
• ?-lactam inhibitor combinations
• e.g., piperacillin-tazobactam

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ESBL Screening Test

• Disk zone (mm) MIC (µg/ml)
• Cefpodoxime ?17 gt4
• Ceftazidime ?22 gt1
• Cefotaxime ?27 gt1
• Ceftriaxone ?25 gt1
• Aztreonam ?27 gt1

only these drugs appropriate for P.
mirabilis gt1 ?g/ml for P. mirabilis
CLSI M100-S15 Table 2A (M2, M7)
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ESBL Phenotypic Confirmatory Test

• Test
• cefotaxime
• cefotaxime/clavulanic acid
• ceftazidime
• ceftazidime/clavulanic acid
• Results
• clavulanic acid restores activity of cefotaxime
  or ceftazidime or both
• QC
• E. coli ATCC 25922 K. pneumoniae ATCC 700603

CLSI M100-S15 Table 2A (M2, M7)
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ESBL Confirmatory Test Positive for ESBL
Cefotax/CA
Ceftaz/CA
Ceftaz
Cefotax
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ESBL Confirmatory Test Negative for ESBL
Ceftaz/CA
Cefotax/CA
Ceftaz
Cefotax
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E-test for detection of ESBLs

• AB Biodisk (Solna,Sweden) has introduced a two
  sided ESBL E-test strip that contain either a
  combination of ceftazidime and ceftazidime/clavula
  ni acid or cefotaxime and cefotaxime /clavulanic
  acid

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E-test for detection ESBls

• Both strips have a decreasing gradient of
  ceftazidime or cefotaxime alone on one end and a
  decreasing gradient of ceftazidime or cefotaxime
  plus a fixed gradient of clavulanic acid on the
  other end .Agt3log reduction in the MIC of
  cefotaxime or ceftazidime in the presence of the
  clavulanicis considered a positive.

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ESBL Confirmatory Test
Ceftaz/CA
Ceftaz
Etest
MicroScan ESBL panel Vitek confirmatory test
Vitek 2 - Advanced Expert
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E. coli - probable AmpC (plasmid-mediated)
MIC (µg/ml)

• ampicillin gt 32 R
• cefazolin gt 32 R
• cefotaxime gt 32 R
• cefoxitin gt 32 R
• cefepime 8 S
• ciprofloxacin 0.5 S
• imipenem ? 0.25 S
• pip/tazo 128 R

Confirmatory tests for E. coli indicate unusual
resistance (but NOT due to ESBLs)
Ucla example
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If ESBL screen test is positive, must we do the
confirmatory test prior to reporting isolate as
an ESBL?

• Yes - unless you are confident that the isolate
  is / is not an ESBL producer based on
• A previous isolate from the patient
• An endemic strain in your institution

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What about testing / reporting for ESBLs on urine
isolates?

• The suggestion (CLSI M100-S15)
• The decision to perform ESBL screening tests on
  all urine isolates should be made on an
  institutional basis.
• Rationale
• ?-lactam concentration in bladder is very high
  and overcomes hydrolysis by ESBL enzymes
• The concerns
• Identifying urine from acute cystitis vs. other
• Patient with isolate from urine other sites
• Infection control issues

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Will CLSI confirmatory test detect ALL
ESBL-producing GNR?

• No - some isolates have ESBLs plus other
  resistance mechanisms that mask ESBL detection in
  the confirmatory test, e.g.,
• gt 1 ESBL
• ESBL ampC
• ESBL porin mutation
• ESBLs occur in species other than E. coli,
  Klebsiella spp., and Proteus mirabilis which CLSI
  does not currently address

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Current CLSI Efforts Re ESBLs

• Determine best way to detect isolates with all
  different types of ESBLs or other ?-lactam
  resistance mechanisms
• Cephalosporin and penicillin breakpoints MAY be
  lowered
• Example cefotaxime
• Current Susceptible at ? 8 ?g/ml
• Proposed Susceptible at ? 1 or ? 2 ?g/ml

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UCLA Inpatient Non-urineIsolates (2000-2004)
of total tested
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ESBL Testing E. coli and Klebsiella spp.
Strategy

• Perform screen test (especially isolates from
  normally sterile sites)
• Perform confirmatory test or send to reference
  lab
• (P. mirabilis perform screen and
  confirmatory test on isolates from sterile sites)

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Proteus mirabilis ESBL testing

• Routine screening not recommended
• Perform ESBL test when clinically relevant (e.g.
  bcteremia)
• -consult with medical staff to determine to test
• -Practical approach ,test sterile body site
  isolate
• Use Standard ESBLs screen test with slight
  modification

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Proteus mirablis ESBLScreen Test

unique breakpoint for P.mirabilis (as compared
to 8 µg /ml for E.coli and Klebsiella spp .)
Naapplicable
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Proteus mirabilis ESBLs Testing (cont)

• Use standard disk diffusion or MIC ESBL
  phenotype confirmatory test without modification

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Proteus mirabilis producer

• Uncommon in USA
• - schwaber et al JCM 2004 .42294
• . 1/171 ESBL positive
• .TEM ß-lactamase gene
• ? Have caused significant problems in some
  locations (e.g. Europe, south America)
• -Nosocomial outbreaks

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ESBLs Test QC frequency Disk Diffusion and MIC
Methods
ESBLs gene on plasmid must store QC strain
at-60C or lower
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Microbiology Laboratories and ESBLs

• Unfortunately ,many clinical laboratories lack
  of understanding regarding ESBLs and Ampc
  ß-lactamase and their detection .This has been
  documented in a study in Connecticut USA, where
  it was found that 21 of laboratories failed to
  detect extended spectrum cephalosporins and
  Aztreonam in ESBLs and Ampc.

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Prevalence of ESBLs

• The true prevalence of ESBLs is not known and
  is probably underestimated because of
  difficulties encounter in their detection.
  However ,it is clear that ESBLs producing
  organisms are distributed worldwide and their
  prevalence is increasing.

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ESBls in Europe

• ESBLs were first described in 1983 from Germany
  and England.
• In Netherlands less than 1 of E.coli and
  K.pneumoniae were ESBLs In France and Italy
  more than 40 strains of K.pneumoniae resistant
  to Ceftazidime.
• It is not known why the prevalence varies so
  widely in closely related regions.

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North America

• The first ESBLs producing organisms reported in
  1988..The prevalence of ESBLs production among
  Enterobacteriaceae in the USA ranges from 0 to 25
  with the national average being 3

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Prevalence of ESBLs USA (cont, )

• CDC Report from ICUs(1999)
• -E.coli 48
• -K.pneumoniae 10.4

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Latin America

• ESBLs appear common in many Latin Americans
  countries.
• The SENETRY study surveillance program among
  10,000 bacterial from ten centers showed
• -45 K.pneumoniae ESBLs positive
• - 8.5 E.coli ESBLs positive

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Asia and Australia

• The prevalence of ESBL producing strains of
  Enterobacteriaceae varies fro country to country
  and from species to species'.
• - E.coli 5 in Korea and 23.3 in Indonesia
  .
• - Klebsiella spp 48.8 in Korea and 20-40
• Southeast Asia , China and Japan .Outbreaks
  of infections due to ESBls producing organisms
  have been described widely in Australia

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Africa and Middle East

• Although national surveillance are not lacking
  ,outbreaks of infections due to ESBL-producing
  organism have bee noted in some African nations,

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Iran

• There is not published data due to ESBLS in
  Iran.
• An study carried out by Rastgari et all in
  enterobacteriacae . Of 171 isolates from urine
• 6 strains of E.coli were ESBLs
• 11 strains of K.pneumoniae were ESBLs

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Infection Control caused by ESBLs

• Isolation measure
• - private room, Private bathroom
• - Contact isolation (gloves .gown.
• - Hand hygiene compliance
• - Dedicated equipment (Thermometer,
  Stethoscope)

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Infection control(cont,)

• Identification of patients
• -patients charts should be flagged so that
  contact isolation for ESBL-colonization can be
  considered on subsequent admissions thorough
  decontamination for environmental of care with
  routine low-level disinfectant (Quaternary
  ammonium ,phenol)

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Infection Control(cont,)

• Antibiotic Use
• Antibiotic use protocol should be institute
  eliminate indiscriminate use of antibacterials
  and decrease selective pressure for ESBLs
  producing strains of bacteria

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Thank you!