1. Extended-Spectrum -lactamases: Current situation, Diagnosis & Management
Siriluck Anunnatsiri, MD
Infectious Diseases & Tropical Medicine
Faculty of Medicine
Khon Kaen University

2. Extended-spectrum -lactamases
Mutant, plasmid-mediated -lactamases enzymes derived from amino acid substitutions in native -lactamases, particularly TEM-1, TEM-2, and SHV-1
Their ability permit hydrolyze all penicillins, cephalosporins (except cephamycins), and monobactams
Typically associated with multidrug resistance (fluoroquinolones, co-trimoxazole, aminoglycosides)
Cephamycins มี 7-methoxy side chain ซึ่งสามารถ block hydrolyze จาก class A & D beta-lactamases.
Oxyimino-beta-lactams มี oxyimino side side chain เป็นตัวจับกับ beta-lactamases โดยทั่วไปหมายถึง 3rd generation cephalosporins รวม 4th generation cephalosporin ด้วย

3. Extended-spectrum -lactamases
Most commonly produced by Klebsiella spp., Escherichia coli but can occur in other GNB, including Enterobacter, Salmonella, Proteus, and Citrobacter spp., Morganella morganii, Serratia marcescens, Shigella dysenteriae, Pseudomonas aeruginosa, and Burkholderia cepacia

4. Major groups of -lactamases
Functional group
Major subgroup
Molecular class
Inhibition by clavulanate 1 C
Cephalosporinases, often chromosomal enzymes in GNB but may be plasmid-encoded, confer resistance to all classes of -lactams, except carbapenems (unless combine with porin change) - 2 2a A
Penicillinases, confer resistance to all penicillins, primarily from Staphylococcus and enterococci + 2b
Broad-spectrum -lactamases (penicillinases/cephalosporinases) , primarily from GNB.
2be
ESBLs, confer resistance to oxyimino-cephalosporins and monobactams.
2br
Inhibitor-resistant TEM (IRT) -lactamases
- (+ for tazobactam) 2c
Carbenicillin-hydrolyzing enzymes
Functional classification (Bush-Jacoby-Medeiros: 1-4) – spectrum of ATB substrate profile, enzyme inhibitor profile, enzyme net charge, hydrolysis rate, binding affinity, isoelectric focusing, protein molecular weight, amino acid composition
Molecular classification (Ambler’s classification: A-D) – nucleotide and amino acid sequence
Shah AA, et al. Research in Microbiology 2004; 155:

5. Major groups of -lactamases
Functional group
Major subgroup
Molecular class
Inhibition by clavulanate 2 2d D
Cloxacillin- (oxacillin)- hydrolyzing enzymes
+/- 2e A
Cephalosporinases, confer resistance to monobactams + 2f
Carbapenem-hydrolyzing enzymes with active site serine (serine based carbapenemases) 3
3a, 3b, 3c B
Metallo--lactamases (zinc based carbapenemases), confer resistance to carbapenems and all -lactam classes, except monobactams. - 4
Miscellaneous unsequenced enzymes that do not fit into other groups
Functional group classified by Bush-Jacoby-Medeiros.
Molecular group classified by Ambler.
Functional classification (Bush-Jacoby-Medeiros: 1-4) – spectrum of ATB substrate profile, enzyme inhibitor profile, enzyme net charge, hydrolysis rate, binding affinity, isoelectric focusing, protein molecular weight, amino acid composition
Molecular classification (Ambler’s classification: A-D) – nucleotide and amino acid sequence
Shah AA, et al. Research in Microbiology 2004; 155:

6. Selected -lactamases of gram-negative bacteria
Examples
Substrates
Inhibition by clavulanate*
Ambler’s class / Bush’s class
Broad-spectrum
TEM-1, TEM-2, SHV-1
Penicillin G, aminopenicillins,
carboxypenicillins, piperacillin, narrow-spectrum cephalosporins
+++
A / 2b
OXA family
Broad-spectrum group plus cloxacillin, methicillin, and oxacillin +
D / 2d
Extended-spectrum
TEM family, SHV family
Broad-spectrum group plus oxyimino-cephalosporins, and monobactam (aztreonam)
++++
A / 2be
CTX-M family
Expanded-spectrum group plus, for some enzymes, cefepime A
Same as for CTX-M family
Others (PER-1, PER-2,
BES-1, GES/IBC family,
SFO-1, TLA-1, VEB-1, VEB2)
Same as for TEM family and SHV family
*+, +++ , and ++++ denote relative sensitivity to inhibition.
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

7. Selected -lactamases of gram-negative bacteria
Examples
Substrates
Inhibition by clavulanate*
Ambler’s class/ Bush’s class
AmpC
ACC-1, ACT-1, CFE-1,
CMY family, DHA-2, FOX
family, LAT family, MIR-1,
MOX-1, MOX-2
Expanded-spectrum group plus
cephamycins
C / 1
Carbapenemase
IMP family, VIM family,
GIM-1, SPM-1 (metallo-enzymes)
cephamycins and carbapenems
B / 3
KPC-1, KPC-2, KPC-3
Same as for IMP family, VIM family, GIM-1, and SPM-1
+++
A / 2f
OXA-23, OXA-24, OXA-25,
OXA-26, OXA-27, OXA-40,
OXA-48 +
D / 2d
*+, +++ , and ++++ denote relative sensitivity to inhibition.
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

8. Major sources of extended-spectrum -lactamases
Type
Major sources
TEM, SHV
E. coli, K. pneumoniae
Cefotaxime hydrolyzing (CTX-M)
S. Typhimurium, E. coli, K. pneumoniae
Oxacillin hydrolyzing (OXA)
P. aeruginosa
PER-1
PER-2
P. aeruginosa, A. baumanii, S. Typhimurium
S. Typhimurium
VEB-1
E. coli, P. aeruginosa

9. Prevalence of ESBL-producing isolates in Europe (1997-2004) and USA (1999-2004)
Goossens H, Grabein B. Diagn Microbiol Infect Dis 2005; 53:

10. Paterson DL et al. J Antimicrob Chemother 2005;55:965-73.
Distribution of ESBL in E. coli SMART, 2003, IAI, Asia-Pacific Countries %
<48 hours
48 hours
Paterson DL et al. J Antimicrob Chemother 2005;55:

11. Paterson DL et al. J Antimicrob Chemother 2005;55:965-73.
Distribution of ESBL in Klebsiella spp. SMART, 2003, IAI, Asia-Pacific Countries %
<48 hours
48 hours
The similar situation is also found in Klebsiella species.
Paterson DL et al. J Antimicrob Chemother 2005;55:

12. Prevalence of ESBL producing organisms 346 isolates of GNB, Siriraj Hospital, 2003
Chayakulkeeree M, et al. Southeast Asian J Trop Med Public Health 2005; 36:

13. Prevalence of ESBL-producing organisms 2974 isolates of GNB, Srinagarind Hospital, 2005%

14. Risk factors associated with infection or colonization with ESBL-producing pathogens
Critically ill patients / Severely debilitated residents
Prolonged hospital or ICU unit stay
Invasive procedures:
indwelling catheter, central venous catheter, gastrostomy, tracheostomy, endotracheal or nasogastric tube
Residency in long-term care facility
Decubitus ulcer
Total dependence on health care workers
Prior antibiotic use in last 3 months
Exposure to 2nd-3rd cephalosporins, aztreonam, penicillins, and quinolones
Delayed appropriate therapy

16. Disk diffusion zone (mm)
CLSI screening criteria for ESBLs in K. pneumoniae, K. oxytoca, and E.coli
Antimicrobial agents
Disk diffusion zone (mm)
MIC (/g/ml)
Cefpodoxime
<17
>8
Ceftazidime
<22
>2
Aztreonam
<27
Cefotaxime
Ceftriaxone
<25
Normal interpretation
Susceptibility zone for E.coli & Kleibsella: ceftazidime 18 mm, cefotaxime 23 mm, ceftriaxone 21 mm
Stürenburg E, Mark D. J Infect 2003; 47:

17. Laboratory tests for ESBLs detection
Method
Presence of ESBLs if..
Double disk approximation or double disk synergy
Disk of 3rd cephalosporin placed 30 mm from amoxicillin-clavulanic acid
Enhanced inhibition
Combination disk
Uses 2 disks of 3rd cephalosporin alone and combined with clavulanic acid
An increase of >5 mm in zone inhibition with use of the combination disk
Microdilution
A broth containing 1 g/mL 3rd cephalosporin
Presence of growth

18. Laboratory tests for ESBLs detection
Method
Presence of ESBLs if..
MIC broth dilution
MIC of 3rd cephalosporin alone and combined with clavulanic acid
A decrease in the MIC of the combination of >3 log2 dilutions
E-test (MIC ESBL strips)
Two-sided strip containing ceftazidime on one side and ceftazidime-clavulanic acid on the other
MIC ceftazidime > 8
MIC combination
Phantom zone
Automated instruments (e.g., Vitek)
Measures MICs and compares the growth of bacteria in presence of ceftazidime vs. ceftazidime-clavulanic acid
Molecular (DNA probes, PCR, RFLP)
Targets specific nucleotide sequences to detect different variants of TEM and SHV genes

19. Confirmatory tests for ESBL detection
รพ. ศรีนครินทร์ ใช้วิธ๊ combined disk test ใช้ยา 3 ตัวคือ ceftazidime, cefotaxime, ceftriaxone
Stürenburg E, Mark D. J Infect 2003; 47:

20. Multi-drug resistance in ESBL-producing organisms
Chayakulkeeree M, et al. Southeast Asian J Trop Med Public Health 2005; 36:

21. Resistance of ESBL-Producing E. coli and K
Resistance of ESBL-Producing E. coli and K. pneumoniae 1,182 Isolates, Srinagarind Hospital, Khon Kaen 2005 %

22. Resistance of ESBL-Producing E. coli and K
Resistance of ESBL-Producing E. coli and K. pneumoniae 1,182 Isolates, Srinagarind Hospital, Khon Kaen 2005 %

23. Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients
Independent risk factors for hospital mortality
Risks
Adjusted OR
95%CI
Inadequate antimicrobial therapy
4.26
3.35–5.44
Acquired organ system derangements (1-organ increment)
3.25
Use of vasopressors
2.20
Underlying malignancy
1.81
APACHE II score
1.05
Increasing age (1-year increment)
1.02
Surgical patient
0.40
Kollef MH et al. Chest 1999; 115:

24. Risk factors for IIAT Retrospective study, 32/187 (17%) patients died
Inadequate initial antimicrobial therapy (IIAT) was a risk factor for mortality
OR 10.04, 95% CI ( )
Risk factors for IIAT
Infection with multidrug-resistant ESBLs (14.58 [ ])
Health care-acquired ESBLs infection (4.32 [ ])
Arch Intern Med 2005;165:

25. Experienced failure of cephalosporin therapy
Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing ESBL
MIC
(g/mL)
Patients, % (n)
Experienced failure of
cephalosporin therapy
Died of bacteremia
within 14 Days 8
100 (6/6)
33 (2/6) 4
67 (2/3)
0 (0/3) 2
33 (1/3)
<1
27 (3/11)
18 (2/11)
Total*
54 (15/28)
MIC = minimum inhibitory concentration
*Includes 5 patients with isolates for which MICs were recorded simply as 0.5 to 4 mg/L.
การศึกษาในผู้ป่วย K. pneumoniae bacteremia
Peterson et al. J Clin Microbiol 2001; 39:

26. Clinical implications of ESBL-producing Klebsiella species and Escherichia coli on cefepime effectiveness
A retrospective, case–controlled study
None-urine source (~80% from lung) of 10 ESBL-cases & 20 controls (non-ESBL) treated with cefepime (2 grams/day, adjusted for GFR)
Risk estimates for the effect of ESBL presence on cefepime outcomes
Variables
OR (95%CI)
Unsuccessful clinical response
9.7 ( )
Unsuccessful microbiological response
28.5 ( )
All-cause mortality
2.0 ( )
Infection-related mortality
4.7 ( )
Kotapati S, et al. J Infect 2005; 51:

27. Cefepime Carbapenems P-value Clinical improvement 62% 70% 0.59
High-dose cefepime as an alternative treatment for infections caused by TEM-24 ESBL-producing Enterobacter aerogenes in severely-ill patients
Retrospective study
Seriously-ill patients infected with ESBL-producing Enterobacter aerogenes, mostly TEM-24
21 treated with cefepime (6 grams/day) / 23 treated with carbapenems (in combination with ciprofloxacin or amikacin)
Cefepime Carbapenems P-value
Clinical improvement 62% 70%
Bacteriological eradication 14% 22%
30-day mortality rate 33% 26%
Nevertheless, a statistically significant increase in failure to eradicate ESBL-producing E. aerogenes was observed as the MICs of cefepime rose (p=0.017).
K. Goethaert et al. Clin Microbiol Infect 2006; 12:

28. Cefepime versus Imipenem-Cilastatin for Treatment of Nosocomial Pneumonia in Intensive Care Unit Patients: a Multicenter, Evaluator-Blind, Prospective, Randomized Study
A randomized, evaluator-blind, multicenter tria
Compared cefepime (6 g/day) vs. imipenem-cilastatin (2 g/day) for the treatment of nosocomial pneumonia in 281 intensive care unit patients.
In subgroup analysis, therapy of pneumonia caused by an organism producing an extended spectrum-lactamase failed in 4 of 13 patients (31%) in the cefepime group but in none of 10 patients in the imipenem group.
Zanetti G, et al. Antimicrobe Agents Chemother 2003; 47:

29. ESBL (TEM-52) – K. pneumoniae bacteremia, non-fatal disease
Bacteremia due to Klebsiella pneumoniae isolates producing the TEM-52 extended-spectrum -lactamase: treatment outcome of patients receiving imipenem or ciprofloxacin
Retrospective study
ESBL (TEM-52) – K. pneumoniae bacteremia, non-fatal disease
10 treated with imipenem / 7 treated with ciprofloxacin
Ciprofloxacin* Imipenem P-value
Treatment failure 5/7 2/
* 2/7 = partial response
Because the isolates had MICs of ciprofloxacin close to the susceptibility breakpoint, treatment failure could be ascribed to the inability of the drug to reach therapeutic concentrations at infected sites.
Endimiani et al. Clin Infect Dis 2004; 38:

30. A prospective, observational study 12 centers, 455 episodes
18.7% with ESBL-K. pneumoniae
Failure to treat with active antibiotic during the 5-day period after positive blood culture was associated with higher mortality rate (63.6% vs 14.1%, p=0.001; OR 10.7 (95%CI ))

31. Pharmacodynamics of intermittent infusion piperacillin/tazobactam and cefepime against ESBL-producing organisms
 Cefepime 1 gram q 12 hrs  Pip/tazo 4.5 grams q 8 hrs
 Cefepime 1 gram q 8 hrs  Pip/tazo grams q 6 hrs
 Cefepime 2 gram q 12 hrs  Pip/tazo grams q 4 hrs
Reese AM, et al. Int J Antimicrobe Agents 2005; 26:

32. Pharmacodynamics of continuous infusion piperacillin/tazobactam and cefepime against ESBL-producing organisms
 Cefepime 3 grams  Pip/tazo 6.75 grams
 Cefepime 4 grams  Pip/tazo 13.5 grams
Reese AM, et al. Int J Antimicrobe Agents 2005; 26:

33. Pharmacodynamics of levofloxacin, gatifloxacin, and ciprofloxacin against ESBL-, and non-ESBL producing organisms
Regimen
Probability (%) of achieving a free AUC/MIC > 125
Non-ESBL producers (n=45)
ESBL producers (n=39)
Levofloxacin 500 mg q 24 hr 88 11
Levofloxacin 750 mg q 24 hr 91 13
Gatifloxacin 400 mg q 24 hr 85 8
Ciprofloxacin 400 mg q 12 hr 2
Moczygemba LR, et al. Clin Ther 2004; 26:

34. Summary of 3rd-generation cephalosporins on treatment of ESBL-producing organisms
Clinical significance of inoculum effect
Poor clinical outcomes are observed when 3rd-generation cephalosporins are used for treatment
Higher fatal outcome
Higher rate of clinical failure
3rd-generation cephalosporins should not be used to treat serious infections with ESBL-producing organisms, even in the presence of apparent susceptibility.
Peterson et al. J Clin Microbiol 2001; 39:
Ariffin H et al. Int J Infect Dis 2000; 4: 21-5.
Wong-Beringer et al. Clin Infect Dis 2002; 34:

35. Summary of 4th-generation cephalosporins on treatment of ESBL-producing organisms
More stable than 3rd-generation cephalosporins againt some ESBLs and very stable against AmpC-type -lactamases
Inoculum effect, susceptible to SHV-type
Need high dosage (> 4 grams/day) of cefepime for achieving the T>MIC target, preferably in combination with aminoglycoside for synergistic effect
Cefepime should not be used to treat serious infections with ESBL-producing organisms.

36. Summary of -lactam/-lactamase inhibitor on treatment of ESBL-producing organisms
Limited clinical information
Class A ESBLs are susceptible to clavulanate and tazobactam in vitro, nevertheless many producers are resistant to -lactamase inhibitor due to
Hyperproduction of the ESBLs → overwhelm inhibitor
Co-production of inhibitor-resistant penicillinases (e.g. OXA-1) or AmpC enzyme
Relative impermeability of the host strain
-lactam/-lactamase inhibitor should not be used to treat serious infections with ESBL-producing organisms.

37. Summary of cephamycins on treatment of ESBL-producing organisms
Limited clinical data
Generally effective against Enterobacteriaceae producing TEM-, SHV-, and CTX-M-derived ESBLs
Cefotetan > cefoxitin : lower MICs
Reports of cephamycins resistance development during prolonged therapy
Loss of outer membrane porin (porin deficient mutant)
Acquisition of plasmid-mediated AmpC -lactamase (ACT-1)

38. Summary of treatment recommendations for infections with ESBL producers
No treatment
Colonization with ESBL producers
Imipenem or meropenem
Bloodstream infection
Ventilator-associated pneumonia
Any producers that appear to have reduced susceptibility to ertapenem
Ertapenem
Complicated urinary tract infections
Intra-abdominal infections
Diabetic food infections
Quinolones
Infections in patients with risk for allergy to carbapenems, if isolates are susceptible
Nitrofurantoin or fosfomycin
Uncomplicated lower urinary tract infection
Tigecycline, colistin, or polymyxin B
Isolates resistant to all other antibiotic options
Patients allergic to -lactams
Livermore DM, Peterson DL. ESBLs in resistance 2006.

39. Carbapenem classification
GROUP 1
Carbapenems
(community acquired infections)
GROUP 2
(hospital acquired infections – pseudomonas activity)
GROUP 3
(hospital acquired infections –
Pseudomonas and MRSA activity)
Ertapenem
Imipenem
Meropenem
Doripenem
Panipenem
CS-023

40. Dosage and Cost of Treatment in Patients with ESBL-producing bacteria Infections
Dose / Day
Cost / Unit
(Baht)
Cost / Day
Ertapenem
1 gm OD
1,735.00
Imipenem/Cilastatin
0.5 gm q 6 hr
750.00
3,000.00
Meropenem
1 gm q 8 hr
1,390.00
4,170.00
Choice of Ertapenem can save cost of treatment about 1,265-2,435 Baht in patients with ESBL-producing bacteria infections.
* ราคายาโรงพยาบาลศรีนครินทร์ ณ วันที่ 21 มิถุนายน พ.ศ. 2549

41. Control of a Prolonged Outbreak of ESBL-Producing Enterobacteriaceae in a University Hospital
■ Imported ESBL-cases
 Acquired ESBL-cases
Lucet JC, et al. Clin Infect Dis 1999; 29:

42. Class restriction of cephalosporin use to control total cephalosporin resistance in nosocomial Klebsiella
Kg drug/month
1995
1996
Total cephalosporins
3rd-generation cephalosporins
Cefuroxime
Cefotetan
Cephazolin
Imipenem
No. cephalosporin-resistant Klebsiella
No. imipenem-resistant Pseudomonas
5.6
0.78
2.2
1.5
1.4
0.2
150 67
1.1
0.39
0.19
0.06
0.44
0.47
84*
113*
* P < 0.01
Rahal JJ, et al. JAMA 1998; 280:

43. Piperacillin/Tazobactam gr.
OASIS I : Bowel Colonization with resistant GNB after antimicrobial therapy of IAI
Organism recovered
from assessable
patients a
Ertapenem gr.
Piperacillin/Tazobactam gr.
Baseline
(n= 162)
End of
therapy
(n= 155)
2 weeks
post-
(n= 133)
(n= 160)
(n= 156)
Piperacillin/Tazo-resistant
Enterobacteriaceae, n (%) 1
(0.6) 2
(1.3) 3
(2.3) 19
(12.2) 6
(4.5)
Ertapenem-resistant
(0)
(1.9)
(0.8)
ESBL-producing
Enterobacteriaceae, n (%) 4
(2.6)
Imipenem-resistant
P. aeruginosa, n (%)
DiNubile MJ et al. Eur J Clin Microbiol Infect Dis 2005; 24:

44. Ceftriaxone/Metronidazole
OASIS II : Bowel Colonization with resistant GNB after antimicrobial therapy of IAI
Organism recovered from assessable patients a
Ertapenem
Ceftriaxone/Metronidazole
Baseline
(n= 201)
End of
therapy
(n= 196)
2 weeks
post-
(n= 182)
Baseline (n= 195)
(n= 193)
(n= 174)
Ceftriaxone-resistant
Enterobacteriaceae, n (%) 9
(4.5) 3
(1.5) 5
(2.7)
(2.6) 33
(17.1) 39
(22.4)
Ertapenem-resistant 1
(0.5)
(0)
ESBL-producing
Enterobacteriaceae, n (%) 8
(4.0) 4
(2.2)
(2.1) 18
(9.3) 30
(17.2)
Imipenem-resistant
P. aeruginosa, n (%) 2
(1.0)
DiNubile MJ et al. Eur J Clin Microbiol Infect Dis 2005; 24:

45. OASIS I-II : Bowel Colonization with resistant GNB after antimicrobial therapy of IAI
ESBL-producing Enterobacteriaceae
DiNubile MJ et al. Eur J Clin Microbiol Infect Dis 2005; 24:

46. OASIS I-II : Bowel Colonization with resistant GNB after antimicrobial therapy of IAI
P. aeruginosa resistance
DiNubile MJ et al. Eur J Clin Microbiol Infect Dis 2005; 24:

47. Individual patient level
Summary of interventions that could be used to prevent problem with ESBL-producing bacteria in hospitalized patients
Individual patient level
Avoid use of 3rd-generation cephalosporins, aztreonam, or cefuroxime
Avoid unnecessary use of invasive devices
Ensure good hand hygiene before and after patient-care activities
Institutional level
Restrict use of 3rd-generation cephalosporins
Introduce contact isolation precautions for patients documented to have carriage or infection with ESBL-producing organisms
Investigate envirinmental contamination if increased rates of ESBL-producing organisms occur
Livermore DM, Peterson DL. ESBLs in resistance 2006.

48. Take Home Messages
ESBL-producing bacterial infection is an emerging problem worldwide.
These organisms are associated with multi-drug resistance causing high rate of mortality and treatment failure.
The significant risk factors for ESBL-producing bacterial infection are prior use of antibiotics, especially 3rd generation cephalosporins, and critically ill or debilitated patients.
Need the ESBL-laboratory testing for establish the problem.
Carbapenems is the drug of choice for serious ESBL-producing bacterial infection.
Avoiding overuse or misuse of 3rd generation cephalosporins and implementing isolation and contact precaution to prevent and control the ESBL outbreak.

49. Thank You for your attention