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Transcript of Esbl
Extended Spectrum β-Lactamases:
Challenges in Laboratory Detection and Implications on
Therapy
Dr. Iman M. FawzyClinical Pathology MD, PhD
Mansoura, Egypt
ESBL
Extended spectrum β-lactamase (ESBL)-producing organisms pose unique challenges to
clinical microbiologists, clinicians, infection control professionals and antibacterial-discovery scientists.
Why we need esbl detection?
• ESBL-producing Enterobacteriaceae have been responsible for numerous outbreaks of infection throughout the world
• ESBL pose challenging infection control issues.
• ESBLs are clinically significant and indicate the appropriate antibacterial agents.
• Unfortunately, the laboratory detection of ESBLs can be complex and, at times, misleading.
– Penicillin– Cephalosporin– Monobactam– Carbapenem
β-lactam antibiotics
β lactamases
Beta lactamases are enzymes produced by some bacteria that hydrolyze beta lactam antibiotics
– Penicillinases, Cephalosporinases– Extended spectrum β-lactamases (ESBL)– Metallo β lactamases– Amp C– Carbapenemase
Definition of ESBL• ESBLs are enzymes
– hydrolyzing most penicillins and cephalosporins, and monobactam (aztreonam).
– but not cephamycins and carbapenems
– Susciptable to β-lactamase inhibitors (clavulanate, sulbactam and tazobactam)
Clinical significance
• ESBLs destroy cephalosporins, main hospital antibiotics, given as first-line agents to many severely-ill patients, including those with intra-abdominal infections, community-acquired pneumonias and bacteraemias.
• Delayed recognition inappropriate treatment of severe infections caused by ESBL producers with cephalosporins ↑↑mortality .
Clinical significance
• ESBL-mediated resistance is not always obvious to all cephalosporins in vitro.
• Many ESBL producers are multi-resistant to non-β-lactam antibiotics such as quinolones, aminoglycosides and trimethoprim, narrowing treatment options.
Spread
• direct and indirect contact
• with colonized/infected patients and
• contaminated environmental surfaces. • ESBLs are most commonly spread via unwashed
hands of health care providers.
Risk factors• Critically ill patients, Immunosuppression• Prolonged hospital or ICU unit stay • Invasive procedures: intubation, mechanical
ventillation, catheter • Long-term dialysis within 30 days • Family member with multidrug-resistant pathogens • Prior antibiotic use in last 3 months• High frequency of antibiotic resistance in the
community or in the specific hospital unit• Patient who previously had an antibiotic-resistant
organism (e.g., MRSA, VRE)
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 +
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.
Major groups of -lactamases
Selected -lactamases of gram-negative bacteria-lactamase
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
OXA family Same as for CTX-M family
+ D / 2d
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
++++ A
*+, +++ , and ++++ denote relative sensitivity to inhibition.
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.
Selected -lactamases of gram-negative bacteria-
lactamase
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
0 C / 1
Carbapenemase
IMP family, VIM family,GIM-1, SPM-1 (metallo-enzymes)
Expanded-spectrum group plus cephamycins and carbapenems
0 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
Same as for IMP family, VIM family, GIM-1, and SPM-1
+ D / 2d
*+, +++ , and ++++ denote relative sensitivity to inhibition.
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.
15
• Klebsiella pneumoniae
• Escherichia coli• Proteus mirabilis• Enterobacter cloacae• Non-typhoidal
Salmonella (in some countries)
Common ESBL producers
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-1PER-2
P. aeruginosa, A. baumanii, S. TyphimuriumS. Typhimurium
VEB-1 E. coli, P. aeruginosa
Common ESBL producers
Mechanisms of resistance
• The majority of ESBLs are acquired enzymes, encoded by plasmids.
• Different resistance phenotypes to:– Different expression levels– Different biochemical characteristics such as
activity against specific β-lactams– co-presence of other resistance mechanisms
(other β-lactamases, efflux, altered permeability)
Survival of the fittest
Resistant bacteria survive, susceptible ones die
Mutant emergesslowly
Sensitive cellskilled by antibiotic
Mutant’s progenyoverrun
The Fight
cell
PG
NO LYSIS
The Fight
cell
PG
NO
β-lactamase
cell
PG
NO
β-lactamase
Inhibitor
The Fight
cell
PG
NO
β-lactamase
Inhibitor
LYSIS
The Fight
Sites of infection
UTI55%
Bactremia11%
Skin and soft tis-sue
s12%
Pneumonia
11%
In-tra ab-domi-nal in-fec-tion
s 6%
Others5%
Laboratory Detection of ESBL
Phenotypic MethodsScreeni
ng metho
ds
Genotypic Methods
CLSI 2013
CLSI 2013
Confirmatory methods
• 1- 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 diskDisc with cephalosporin
+ clavulanic acid
Disc with cephalosporin
alone
CLSI 2013
CLSI 2013
Ceftaz/CA
Cefotaxime/CA
Ceftaz
Cefotax
Ceftaz/CA
Ceftaz
Negative ESBL
Positive ESBL
Cefotax
Cefotaxime/CA
Difference > 5 mmCeftaz/CA
Cefotax/CA
Ceftaz Cefotax
Positive ESBL
Ciftazidim + Clav
Cefotaxim + Clav
CeftazidimCefotaxim
Difference > 5 mm
Difference > 5 mm
Difference > 5 mm
2- Double disk approximation or double disk synergy– Disk of 3rd cephalosporin placed 30 mm from amoxicillin-
clavulanic acid
– Result: Enhanced inhibition (A keyhole or ghost zone)
Phenotypic conformation
Amox-clavCefotaxime
Ceftriaxone
Ceftazidime
Azteonam
CeftazidimAugmentin Cefotaxim
CeftazidimeAugmentin
Cefotaxime
AugmentinCefotaxime
Ceftriaxone
AMC AMC AMC
30 mm distance between discs (center to center)
20 mm distance between discs (center to center)
AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; FEP, cefepime; CPO, cefpirome.
30 mm distance between discs (center to center)
20 mm distance between discs (center to center)
AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; FEP, cefepime; CPO, cefpirome.
Phenotypic conformation
• 3- Broth MicrodilutionMIC of 3rd cephalosporin alone and combined with clavulanic acid
>3-two fold serial dilution decrease in MIC of either cephalosporin in the presence of clavulanic acid compared to its MIC when tested alone.
Ceftazidim MIC =8 μg/mL
Ceftazidime + Clavulanate= 1 μg/mL
Or MIC ratio≥8
4- MIC broth dilutionMIC of 3rd cephalosporin alone and combined with clavulanic acid
MIC of 3rd cephalosporin alone and combined with clavulanic acid
A decrease in the MIC of the combination of > 3-two fold dilutions
Phenotypic conformation
• 5- E-test (MIC ESBL strips)• Two-sided strip containing cephalosporin on one side
and cephalosporin -clavulanic acid on the other• MIC ratio ≥8•>8 fold reduction in MIC in presence of CA= ESBL• or Phantom zone (deformed ellipse)
Cefotaxime
Cefotaxime+
clavulanate
Ceftaz/CA
CeftazMIC =16
MIC= 0.25
Other confirmatory methods
No inhibitor
Mercaptoacetic acid to inhibit MBL
Clavulanate to inhibit ESBL
Boronic acid to inhibit AmpC
Cica b-Testuses the chromogenic cephalosporin HMRZ-86,4,5 + inhibitors to determine rapidly whether an isolate has a metallo-β-lactamase (MBL), ESBL, or hyperproduced AmpC enzyme , a control strip with no inhibitor, to detect hydrolysis of extended-spectrum cephalosporins
Other confirmatory methodsBrilliance ESBL agar • identification of ESBL-
producing E. coli, Klebsiella, Enterobacter, Serratia and Citrobacter group, directly from clinical samples.
• two chromogens that specifically target enzymes green and blue colonies
• Negative pink. • Proteus, Morganella and
Providencia tan-coloured colonies with a brown halo
6- Automated instruments• Measure MICs and compare the growth of bacteria in
presence of cephalosporin vs. cephalosporin -clavulanic acid
Other confirmatory methods
Vitek ESBL confirmatory test Phoenix ESBL test (BD)
Microscan ESBL Panel
Genotypic confirmation
• Molecular detection – PCR– RFLP– gene sequencing– DNA microarray-based method
• Targets specific nucleotide sequences to detect different variants of TEM and SHV genes
Control strains
AmpC β-lactamases• third-generation cephalosporins: resistance , • cephamycins, e.g. a cefoxitin: resistance • Cefepime: sensitive.
CarbapenemasesThe presence of ESBLs may also be masked by
carbapenemases
Pitfalls in ESBL tests
ESBLs vs AmpCs
ESBLs AmpCs
Inhibitors (pip/tazo, amp/sulbactam, amox/clav) S R
Cefoxitin, cefotetan S R
Ceftazidime, ceftriaxone R R
Cefepime S/R S
Pitfalls in ESBL tests
ESBL+ AmpC β -lactamases: • Especially in Enterobacter spp., Citrobacter,
Morganella, Providencia and Serratia. • The AmpC enzymes may be induced by
clavulanate (which inhibits them poorly) and may then attack the cephalosporin, masking synergy arising from inhibition of the ESBL.
• Screening criterion for ESBL presence among AmpC producing Enterobacter, C. freundii and ‑Serratia is Cefepime MIC > 1 ug/ml (inhibition
zone< 26 mm).• Use of Cefepime is more reliable to detect
these strains because high AmpC production has little effect on cefepime activity.
Pitfalls in ESBL tests
Amox-Clav
Cefepime
ESBL+ AmpC
ESBL+ AmpC
CefoxitinCefotaxime
Cefipime
Ceftazidime Cefpodoxime
Cefpodoxime + Clavulanic
Augmentin
ESBL+ AmpC
ESBL and AmpC
ESBL positiveclavulanate enhancement present
AmpC positive cefepime: Scefoxitin: R
ESBL+ AmpC
ESBL+ AmpCAmpCFox: RClav: R
ESBLZone enhancement
AmpCcefepime : S cefoxitin : R
no clavulanate enhancement= ESBL negative
AmpC
ESBL + carbapenemases
ESBL positive clavulanate enhancement present carbapenemase production resistance to carbapenem agents
ESBL+ Carbapenemase
ESBLs and the inoculum effect
• In vitro: the MICs of cephalosporins rise as the inoculum of ESBL- producing organisms increases.
• In vivo: Intra-abdominal abscesses and pneumonia are some of the clinical settings where organisms are present in high-inoculum, physicians should avoid cephalosporins if risk of ESBL-producing organism is suspected.
• Two antibiograms of ESBL producing strain. Note the difference in zones and synergistic effect around the amoxicillin-clavulanate pills due to different inoculum concentration.
Reporting
If ESBL: Resistant, for all penicillins, cephalosporins, and monobactams
Report beta lactam inhibitor drugs as they test.
If ESBL is not detected, report drugs as tested.
Treatment
Carbapenems are the drugs of choice.
Unfortunately, use of carbapenems has been associated with the emergence of carbapenem-resistant bacterial species
It may be advisable to use non carbapenem antimicrobials as the first line treatment in the less severe infections with ESBL producing strains.
-lactam/-lactamase inhibitor on treatment of ESBL-producing organisms
• Most ESBLs are susceptible to clavulanate and tazobactam in vitro,
• nevertheless some ESBL producers are resistant to -lactamase inhibitor due to– Hyperproduction of the ESBLs → overwhelm inhibitor– Co-production of inhibitor-resistant penicillinases 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• Reports of cephamycins resistance development
during prolonged therapy– Loss of outer membrane porin (porin deficient
mutant)– Acquisition of plasmid-mediated AmpC -
lactamase (ACT-1)
ESBL are Emerging Challenges
• multiple enzymes • High-Risk clones• globally disseminated• hospital, community acquired• High rates• Challenge of intestinal carriage • extra-human reservoirs
ESBL are more complex
• Antibacterial choice is often complicated by multi-resistance. – Many ESBL producing organisms also express
AmpC β-lactamases – may be co-transferred with plasmids mediating
aminoglycoside resistance. – there is an increasing association between ESBL
production and fluoroquinolone resistance
Prevention
– ICU is hot spot– Hands of healthcare workers, family, visitors– thermometer– Ultrasound gel– Flag records– Education – Contact precautions– Transfer between wards & hospitals
Still the best way to prevent spread of infections and drug resistance is ……
Prevention
Individual patient level
•Avoid use of cephalosporins, aztreonam•Avoid unnecessary use of invasive devices •Ensure good hand hygiene before and after patient-care activities
Institutional level•Restrict use of 3rd-generation cephalosporins•Isolation of patient•Investigate environmental contamination
Recommendations• Older agents such as aminoglycosides need
reappraisal to spare the selective pressures of a carbapenem.
• new trials of cephalosporin/β-lactamase inhibitors can be predicted
• oral carbapenems are urgently needed
Recommendations
• Empirical treatment strategies may need to be re-thought where there is a significant risk.
• Use a carbapenem until the infection has been proved NOT to involve an ESBL producer, then to step down to a narrower- spectrum ab .
Recommendations• Optimize appropriate use of antimicrobials
– The right agent, dose, timing, duration, route
• Help reduce antimicrobial resistance– The combination of effective antimicrobial
supervision and infection control has been shown to limit the emergence and transmission of antimicrobial-resistant bacteria
Dellit TH et al. Clin Infect Dis. 2007;44(2):159–177; . Drew RH. J Manag Care Pharm. 2009;15(2 Suppl):S18–S23; Drew RH et al. Pharmacotherapy. 2009;29(5):593–607.
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.
THANK YOU