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Beta Lactamases &
Extended Spectrum Beta Lactamases
Dr.M.Malathi
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Contents• Introduction• Beta Lactam antibiotics• Beta Lactamases• Classification• Methods of detection of Beta Lactamases• Treatment• Extended Spectrum Beta Lactamases• Epidemiology in India• Resistance patterns• Methods of detection of ESBL• Treatment• Conclusion
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Introduction
• The resistance to beta lactam antibiotics is a great concern worldwide .
• Beta lactamases production is the most common mechanism of drug resistance .
• Continuous mutations in due course have lead to extended profile of resistance – Extended spectrum beta lactamases, AmpC beta lactamases and Metallo beta lactamases.
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Beta lactam antibiotics
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BETA LACTAM - MOA
BACTERICIDAL EFFECT
• Covalent binding of the antibiotic to one or more penicillin sensitive enzymes (PBPs)
• Inhibition of the enzymes responsible for catalyzing peptide cross linking in the biosynthesis of the cell wall.
• No cross linkage between the peptidoglycan precursors
• Increase in internal osmotic pressure of the cell• Lysis of the cell and cell death
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Mechanism of action
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BETA LACTAM RESISTANCE
• Inactivation of the penicillin through B-lactamase- or penicillinase-mediated hydrolysis of the B-lactam ring of the antibiotic.
• Alteration of the target- intrinsic resistance involving a lowering of the affinity or the amount of the PBPs
• Tolerance to the bactericidal effect of B-lactam antibiotics
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BETA LACTAMASE • Kirby first demonstrated that penicillin was
inactivated by penicillin-resistant strains of S. aureus .
• Genes that encode beta lactamase production can be seen in plasmids, chromosomes or in transposons.
• Beta lactamase in S.aureus is an extracellular enzyme - Plasmid mediated.
• The beta lactamase responsible for ampicillin resistance in Klebsiella pneumoniae is in chromosome.
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Types
• Inducible – turned off without drug – plasmid mediated - Eg: Staph aureus
• Constitutive – SHV – 1 chromosomal enzyme of Kleb pneumoniae - responsible for ampicillin resistance
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Mechanism of resistance
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Regulation of resistance
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Organisms tested for beta lactamase
• Staphylococcus aureus• CONS• Enterococci sp.,• Neisseria gonorrhea• Hemophilus influenzae• Moraxella catarrrhalis
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Methods of detection
Phenotypic method:• Acidometric method• Iodometric method• Nitrocefin method• penicillin disc diffusion test• Penicillin broth microdilution test• Penicillin zone edge testGenotypic test:• PCR for blaZ gene
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DETECTION OF BETALACTAMASE
• Difficult to demonstrate the manifestation in vitro• The bacterial concentration needs to be high(>106
cells/ml). Enzyme must be induced• Problems with agar diffusion method and breakpoint
testing • Only feasible method is detection of enzyme
production with biochemical tests• Acidometric method• Iodometric method• Nitrocefin method
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Acidometric method
Filter paper impregnated with penicillin and indicator dye
Bacterial growth applied to the paper
Alteration in the colour of the indicator seen in positive enzyme production
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Iodometric method
Heavy suspension of test org is made in phosphate bufferwith 6g/l of penicillin from overnight culture
0.1ml into microtitre well-37degrees for 1 hr
2 drops of starch solution
One drop of iodine
Loss of blue colour
Positive test
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Nitrocefin method
• Nitrocefin is a chromogenic cephalosporin which changes from yellow to read when the amide bond in beta-lactam ring is hydrolyzed by beta-lactamase. It is sensitive to hydrolysis by all known lactamases produced by Gram-positive and Gram-negative bacteria.
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Nitrocefin disk test
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PENICILLIN DISC DIFFUSION AND BROTH MICRODILUTION FOR STAPH.AUREUS
(CLSI GUIDELINES)
• For disc difusion: Penicillin 10 units SENSITIVE ≥ 29 INTERMEDIATE – N/A RESISTANT ≤28
• For Broth microdilution: Penicillin SENSITIVE ≥ 0.12ug/ml INTERMEDIATE –N/A RESISTANT ≤ 0.25 ug/ml
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Penicillin disc zone edge test
• Penicillin disc diffusion zone edge test:10U disc
• Sharp zone /cliff edge– β lactamase positive
• Fuzzy zone / beach edge- β lactamase negative
• Indication- Negative nitrocephin test Resistant in disc diffusion or broth microdilution
methods
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Penicillin zone interpretation
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CLSI SAYS…
• The penicillin disk diffusion zone-edge test was shown to be more sensitive than nitrocefin-based tests for detection of β-lactamase production in S. aureus.
• “The penicillin zone-edge test is recommended if only one test is used for β-lactamase detection. However, some laboratories may choose to perform a nitrocefin-based test first and, if this test is positive, report the results as positive for β-lactamase (or penicillin resistant). If the nitrocefin test is negative, the penicillin zone-edge test should be performed before reporting the isolate as penicillin susceptible in cases where penicillin may be used for therapy .eg:endocarditis”
• β-lactamase–positive staphylococci are resistant to penicillin, amino-, carboxy-, and ureidopenicillins.
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Interpretation
• A positive beta lactamase production means that the test organism is resistant to following antibiotics:
1. Penicillin2. Amoxycillin3. Ampicillin4. Piperacillin5. Mezlocillin6. Carbenicillin
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TREATMENT
• Beta lactamase resistant semisynthetic penicillin( methicillin, nafcillin, cloxacillin,dicloxacillin)
• Beta lactam – betalactamase inhibitor combinations ( eg: amoxicillin clavulunate, Ampicillin sulbactam, Piperacillin tazobactam)
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Extended Spectrum Beta Lactamases
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Definition
ESBLs are beta lactamases capable of conferring bacterial resistance to the penicillins, first-, second-, and third-generation cephalosporins and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, and which are inhibited by beta lactamase inhibitors such as clavulanic acid
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Epidemiology
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Diversity of ESBL types
• SHV• TEM• CTX – M • Toho beta lactamases• PER
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SHV
• This type of ESBL is the most frequently isolated one
• SHV - Sulfhydryl variable• 1983, Klebsiella ozaenae isolated from
Germany – possesed a beta lactamase which efficiently hydrolysed cefotaxime and to lesser extent ceftazidime – Different from SHV – named as SHV -2
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TEM
• TEM 1 – first reported in 1965 – Escherichia coli – from a patient named Temoneira
• TEM 1 – hydrolyse ampicillin at a greater rate than carbenicillin, oxacillin or cephalothin and has negligible activity against extended spectrum cephalosporins.
• TEM 1 and TEM 2 has same hydrolytic profile but differs in isoelectric point
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• In 1987, a novel plasmid mediated beta lactamase coined as CTX-1, because of its enhanced activity against cefotaxime – now renamed as TEM-3
• Now, over 100 TEM types have been described.
• Interesting mutants of TEM – hydrolyze 3rd generation cephalosporins, but also demonstrate inhibitor resistance – complex mutants of TEM – TEM AQ.
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CTX –M
• Organisms having CTX-M type of beta lactamases have cefotaxime MICs in the resistant range, while ceftazidime MICs are usually in the apparently susceptible range.
• Same organism may harbour both CTX-M and SHV type of ESBLs which may alter the resistance phenotype.
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Toho β lactamases
• Structurally related to CTX-M type β lactamases.
• First isolated in Toho refers to the Toho university, omoro hospital, Tokyo , where a child was infected with Escherichia coli infection.
• Worldwide, the most common ESBL type is CTX-M ESBL
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PER
• 25% similarity to TEM and SHV type of ESBLs.• First detected in Pseudomonas aeruginosa and
later in Salmonella Typhimurium and Acinetobacter isolates.
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ESBL producing organisms
• Escherichia coli• Klebsiella sp.,• Enterobacter sp.,• Proteus sp.,• Salmonella sp.,
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• ESBLs producing large multiresistance plasmids are more common in Klebsiella sp., than Escherichia coli.
• The importance of ESBL producing Klebsiella sp., is it survives longer than other enteric bacteria on hands and environmental surfaces – leads to cross infection.
• Outbreak – genotypical analysis is must to identify the single clone of genotypically identical organism
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Risk for ESBL infection
• Seriously ill patients • Prolonged hospital stay• Invasive medical devices• Cross infections• Colonizers in medical staffs• Immunocompromised• Prolonged antibiotic intake
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Mode of spread of ESBL
• Ultrasonography coupling gel• Bronschoscopes• Blood pressure cuffs• Glass thermometers• Patients soap• Sink basins• Hands of health care workers• Cockroaches (Vector of ESBL)
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ESBL detection
• Phenotyping
• GenotypingWhy we have to detect?
Detection of ESBL in samples like urine is important as it represents an epidemiological marker of colonisation and therefore a potential threat of transfer to other patients
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PHENOTYPIC METHODS ( CLSI M100 – S24)
• Screening test: Disk diffusion test
• Confirmatory test:Double disk diffusion testBroth microdilution test
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Disk diffusion screening test
• For Escherichia coli and Klebsiella sp.,:1. Cefpodoxime (10µg) ≤17mm2. Cefotaxime (30µg) ≤27mm 3. Ceftriaxone (30µg) ≤25mm4. Ceftazidime (30µg) ≤22mm5. Aztreonam (30µg) ≤27mm
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• For Proteus mirabilis:1. Cefpodoxime (10µg) ≤22mm2. Ceftazidime (30µg) ≤22mm3. Cefotaxime (30µg) ≤27mm
Use of more than one antimicrobial agent for screening improves the sensitivity of ESBL detection.
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Disk diffusion confirmatory test
• Ceftazidime (30µg) and Ceftazidime-clavulanate (30/10µg)
• Cefotaxime (30µg) and Cefotaxime-clavulanate (30/10µg)
Confirmatory testing requires use of both disks
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≥ 5 mm disk zone difference
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Broth microdilution
• Ceftazidime 0.25 – 128 µg/mL and Ceftazidime - clavulanate 0.25/4 – 128/4 µg/mL
• Cefotaxime 0.25 – 64 µg/mL and Cefotaxime - clavulanate 0.25/4 – 64/4 µg/mL
Confirmatory testing requires use of both dilutions
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≥ 3 twofold concentration decrease in MIC
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Quality control for ESBL
• Escherichia coli ATCC 25922 - ≤ 2 mm increase in zone diameter for antimicrobial agent tested in combination with clavulanate vs the zone diameter when tested alone.
• Klebsiella pneumoniae ATCC 700603 - ≥5mm increase in zone diameter of ceftazidime-clavulanate vs ceftazidime alone.
• ≥3mm increase in zone diameter of cefotaxime-clavulanate vs cefotaxime alone.
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Interpretation
• For all confirmed ESBL producing strains
• Report as resistant to all penicillins, cephalosporins and aztreonam
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Other methods
• E test for ESBL• Vitek ESBL cards• Microscan panels• BD Phoenix automated microbiology system• Double disk diffusion test• Agar supplemented with clavulanate• Disk replacement method• Three dimensional test
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GENOTYPIC METHODS
• Pulsed field gel electrophoresis• Polymerase chain reaction• Ribotyping• Plasmid profile analysis• Ligase chain reaction
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Outbreak analysis
1. Identify patients infected with ESBL producing organisms by the use of appropriate detection methods .
2. Identify colonized patients by use of rectal swabs plated onto selective media.
3. Perform molecular epidemiologic analysis of strains from infected or colonized patients
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4. Institute contact isolation precautions, particularly if clonal spread is demonstrated.
5. Institute controls on antibiotic use, particularly if numerous strain types are demonstrated.
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ESBL producers in stool
• Mac Conkey agar supplemented with ceftazimide 4mg/litre
• Nutrient agar supplemented with ceftazidime 2mg/litre, vancomycin 5mg/litre and amphotericin B 1667mg/litre
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Management of outbreak of ESBL
• Contact isolation with use of gloves and gowns when contacting the patient.
• Digestive decontamination by quinolones, colistin, neomycin and tobramycin.
• Nasal spray with povidone iodine as a means of decolonizing the upper respiratory tract.
• Change the infection control procedures.• Change the empirical treatment.
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Summary
• For detection of β lactamases – Penicillin zone edge test with 10 U
• For detection of ESBL – Disk diffusion test with Ceftazidime (30µg) and Ceftazidime-clavulanate (30/10µg) ; Cefotaxime (30µg) and Cefotaxime-clavulanate (30/10µg)
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References
• Mackie and McCarntney Practical microbiology – 14th edition
• David L.Paterson et al.,(2005), Extended spectrum beta lactamases: a clinical update, clinical microbiology review, ASM, oct2005,p657-686
• Performance standards for antimicrobial susceptibility testing: 24th informational supplement – M100-S24