Extended-Spectrum Extended-Spectrum -lactamases: -lactamases: Current situation, Diagnosis & Current situation, Diagnosis &

ManagementManagement

Siriluck Anunnatsiri, MDInfectious Diseases & Tropical Medicine Faculty of MedicineKhon Kaen University

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)

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

Major groups of -lactamases

Functiona

l grou

p

Major

subgroup

Molecular class

Functional group 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 A Broad-spectrum -lactamases (penicillinases/cephalosporinases) , primarily from GNB.

+

2be A ESBLs, confer resistance to oxyimino-cephalosporins and monobactams.

+

2br A Inhibitor-resistant TEM (IRT) -lactamases

- (+ for

tazobactam

)

2c A Carbenicillin-hydrolyzing enzymes +

Shah AA, et al. Research in Microbiology 2004; 155: 409-421.

Major groups of -lactamases

Functiona

l grou

p

Major

subgroup

Molecular class

Functional group Inhibition by clavulanate

2 2d D Cloxacillin- (oxacillin)- hydrolyzing enzymes

+/-

2e A Cephalosporinases, confer resistance to monobactams

+

2f A 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.

Shah AA, et al. Research in Microbiology 2004; 155: 409-421.

Selected -lactamases of gram-negative bacteria

-lactamase

Examples Substrates Inhibition by clavulanate*

Ambler’s class / Bush’s class

-Broadspectrum

- - 1 2TEM , TEM ,-1SHV

Penicillin G, amino penicillins,

carboxypenicillins, piperacillin, narro

- w spectrum cephalosporins

+++ A / 2b

OXA family B - road spectrum gr oup plus cloxacillin

, methicillin, and oxacillin

+ D / 2d

ExtendExtend-ed spe-ed spe

ctrumctrum

TEM family, SH TEM family, SH V family V family

BB - road spectrum gr- road spectrum gr -oup plus oxyimino -oup plus oxyimino

cephalosporins, an cephalosporins, an d monobactam (azt d monobactam (azt

reonam)reonam)

++++++++ AA / 2be/ 2be

- CTX M family- CTX M family EE -xpanded spectru-xpanded spectru m group plus, for s m group plus, for s

ome enzymes, cefe ome enzymes, cefepimepime

++++++++ AA

OXA family OXA family - Same as for CTX M - Same as for CTX Mfamilyfamily

++ DD / 2d/ 2d

- Others (PER 1, - Others (PER 1,- PER 2,- PER 2,

-BES-BES 1, 1, GES/IBC GES/IBC family, family,

- - 1 , 1 ,- - 1 , 1 ,- VEB 1, VEB2)- VEB 1, VEB2)

Same as for TEM fa Same as for TEM fa mily and SHV famil mily and SHV famil

yy

++++++++ AA

* + , +++ , and ++++ denote relative sensitivity to inhibition.

Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

Selected -lactamases of gram-negative bacteria

-lactamas

e

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 - , 1 ,

- -MOX 1, MOX 2

E -xpanded spectr um group plus

cephamycins

0 C / 1

Carbapenemase

IMP family, VIM family,

- - GIM 1, SPM 1 (metallo-enzymes)

E -xpanded spectr um group plus

cephamycins an d carbapenems

0 B / 3

- - 1 2KPC , KPC , K-PC 3

Same as for IMP family, VIM famil

- y, GIM 1, and SP-E1

+++ A / 2f

- - OXA 23, OXA 24,OXA-25,

- - OXA 26, OXA 27,OXA-40,

-OXA 48

Same as for IMP family, VIM famil

- y, GIM 1, and SP-E1

+ D / 2d

* + , +++ , and ++++ denote relative sensitivity to inhibition.

Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

Major sources of 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

Prevalence of ESBL-producing isolates in Europe (1997-2004) and USA (1999-2004)

Europe USA

Goossens H, Grabein B. Diagn Microbiol Infect Dis 2005; 53: 257-64.

Distribution of ESBL in E. coli SMART, 2003, IAI, Asia-Pacific Countries

0

10

20

30

40

50

Australia China Korea Malaysia NewZealand

Philippines Taiwan Thailand

%

<48 hours 48 hours

Paterson DL et al. J Antimicrob Chemother 2005;55:965-73.

Distribution of ESBL in Klebsiella spp. SMART, 2003, IAI, Asia-Pacific Countries

0

10

20

30

40

50

60

Australia China Korea Malaysia NewZealand

Philippines Taiwan Thailand

%

<48 hours

48 hours

Paterson DL et al. J Antimicrob Chemother 2005;55:965-73.

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: 1503-9.

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

26.3

73.7

36.7

63.3

10.7

89.3

4.4

95.6

0

50

100

E. coli K. pneumoniae K. oxytoca P. mirabilis

ESBLstrain Non-ESBL strain%

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

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 >2

Cefotaxime <27 >2

Ceftriaxone <25 >2

Stürenburg E, Mark D. J Infect 2003; 47: 273-95.

Laboratory tests for ESBLs detectionLaboratory tests for ESBLs detection

Tests 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

Laboratory tests for ESBLs detectionLaboratory tests for ESBLs detection

Tests 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

Confirmatory tests for ESBL detection

Stürenburg E, Mark D. J Infect 2003; 47: 273-95.

Multi-drug resistance in ESBL-producing organismsMulti-drug resistance in ESBL-producing organisms

Chayakulkeeree M, et al. Southeast Asian J Trop Med Public Health 2005; 36: 1503-9.

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

15.4

34.5

75.780.4

21.6

53.3

20

58

33

7380.4

56.6

0

20

40

60

80

100

Amikacin Gentamicin Netilmycin Ofloxacin LevofloxacinCiprofloxacin

E. coli K. pneumoniae%

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

84.48891.692.6

48.256.1

32.2

74.1

0.30.2 05.2 0.30.20

20

40

60

80

100

Amoxi/cla Ampi/sul Cefo/sul Pip/taz ImipenemErtapenemMeropenem

E. coli K. pneumoniae%

Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients

RisksRisks AdjuAdjusted sted OROR

95%95%CICI

Inadequate antimicro bial therapy

4.26 33.5–54

4 Acquired organ syste

m derangements (1-o rgan increment)

3.25 2.98-3.54

Use of vasopressors 2.20 1.81-2.66

Underlying malignancy

1.81 1.44-2.27

APACHE II score 1.05 1.04-1.07

Increasing age (1-year increment)

1.02 1.01-1.03

Surgical patient 0.40 0.33-0.49

Independent risk factors for hospital mortality

Kollef MH et al. Chest 1999; 115: 462-74.

• Retrospective study, 32/187 (17%) patients died

• Inadequate initial antimicrobial therapy (IIAT) was a risk factor for mortality

– OR 10.04, 95% CI (1.90-52.96)

• Risk factors for IIAT – Infection with multidru

g-resistant ESBLs (14.58 [1.91-111.36])

– Health care-acquired ESBLs infection (4.32 [1.49-12.54])

Arch Intern Med 2005;165:1375-80.

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) 0 (0/3)

<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.

Peterson et al. J Clin Microbiol 2001; 39: 2206-12.

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 outcomesRisk estimates for the effect of ESBL presence on cefepime outcomes

Variables OR (95%CI)

Unsuccessful clinical response 9.7 (1.4-68.8)

Unsuccessful microbiological response 28.5 (2.6-306.6)

All-cause mortality 2.0 (0.396-10.1)

Infection-related mortality 4.7 (0.38-60.1)

Kotapati S, et al. J Infect 2005; 51: 211-7.

High-dose cefepime as an alternative treatment for infections High-dose cefepime as an alternative treatment for infections caused by TEM-24 ESBL-producing caused by TEM-24 ESBL-producing Enterobacter aerogenesEnterobacter aerogenes in in severely-ill patientsseverely-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% 0.59Bacteriological eradication 14% 22% 0.7630-day mortality rate 33% 26% 0.44

• 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: 56-62.

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

• C ompared cefepime (6 g/ day) vs. -imipenem c ilastatin (2 g/ day) for the treatment of nosoco

mial pneumonia in 281 intensive care unit patients.

• In subgroup analysis , therapy of pn eumonia caused by an organism pro

-ducing an extended spectrum lacta mase 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: 3442-7.

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 2/10 0.03

* 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: 243-51.

•A prospective, observational study•12 centers, 455 episodes •18.7% with ESBL-K. pneumoniae

Pharmacodynamics of intermittent infusion piperacillin/tazobactam and cefepime against ESBL-producing organisms

Reese AM, et al. Int J Antimicrobe Agents 2005; 26: 114-9.

Cefepime 1 gram q 12 hrs Pip/tazo 4.5 grams q 8 hrs Cefepime 1 gram q 8 hrs Pip/tazo 3.375 grams q 6 hrs Cefepime 2 gram q 12 hrs Pip/tazo 3.375 grams q 4 hrs

Pharmacodynamics of continuous infusion piperacillin/tazobactam and cefepime against ESBL-producing organisms

Reese AM, et al. Int J Antimicrobe Agents 2005; 26: 114-9.

Cefepime 3 grams Pip/tazo 6.75 grams

Cefepime 4 grams Pip/tazo 13.5 grams

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 88 2

Moczygemba LR, et al. Clin Ther 2004; 26: 1800-7.

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: 2206-12.Ariffin H et al. Int J Infect Dis 2000; 4: 21-5.Wong-Beringer et al. Clin Infect Dis 2002; 34: 135-46.

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.

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.

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)

Summary of treatment recommendations for infections Summary of treatment recommendations for infections with ESBL producerswith 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.

Carbapenem classification

GROUP 1Carbapenems

(community acquired infections)

GROUP 2Carbapenems

(hospital acquired infections –

pseudomonas activity)

GROUP 3Carbapenems

(hospital acquired infections –

Pseudomonas and MRSA activity)

Ertapenem

Imipenem

Meropenem

Doripenem

Panipenem

CS-023

Dosage and Cost of Treatment in Patients with ESBL-producing bacteria Infections

Dose / Day Cost / Unit

(Baht)

Cost / Day

(Baht)

Ertapenem 1 gm OD 1,735.00 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

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Choice of Ertapenem can save cost of treatment about 1,265-2,435 Choice of Ertapenem can save cost of treatment about 1,265-2,435 Baht in patients with ESBL-producing bacteria infections.Baht in patients with ESBL-producing bacteria infections.

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: 1411-8.

Class restriction of cephalosporin use to control total cephalosporin resistance in nosocomial Klebsiella

Kg drug/monthKg drug/month 19951995 19961996

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: 1233-7.

OASIS I-II : Bowel Colonization with resistant GNB after antimicrobial therapy of IAI

2.5%

0.6%

2.1%

0.0%

2.6%

9.3%

1.6%0.8%

17.2%

0.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

14.0%

16.0%

18.0%

Baseline End of therapy 2 wk post-therapy

ESBL-producingErtapenem

ESBL-producingPip/Taz

ESBL-producingCeftriaxone

ESBL-producing Enterobacteriaceae

DiNubile MJ et al. Eur J Clin Microbiol Infect Dis 2005; 24: 443-9.

OASIS I-II : Bowel Colonization with resistant GNB after antimicrobial therapy of IAI

0%

1.30%

0%

0.60%

1.30%

0.60%

0.30%

0 0%0.0%

0.2%

0.4%

0.6%

0.8%

1.0%

1.2%

1.4%

Baseline End of therapy 2 wk post-therapy

Imipenem-resistantErtapenem

Imipenem-resistantPip/Taz

Pip/Taz-resistantPip/Taz

P. aeruginosa resistance

DiNubile MJ et al. Eur J Clin Microbiol Infect Dis 2005; 24: 443-9.

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.

Take Home MessagesTake 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.