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Introduction to
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β-lactamases
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Irene Galani, PhD
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Infectious Diseases Laboratory,
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Molecular Biology Section,
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4th Department of Internal Medicine,
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Athens University School of Medicine
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egalani@med.uoa.gr
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β-lactamases
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►Beta-lactamases are bacterial enzymes that hydrolyze the beta-
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lactam ring and render the antibiotic inactive before it reaches the
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PBP target.
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Sir Alexander Fleming Discovers Penicillin
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In 1928
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fA mold on a petri dish was
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observed to inhibit growth of
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Staphylococcus bacteria.
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f The active ingredient
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found to be a safe and
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effective bacteria-killing
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British Journal of Experimental Pathology, but
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little attention was paid to his article.
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S. aureus penicillinase
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1945 Nobel Prize Acceptance Speech
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Sir Alexander Fleming warns
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of the danger of resistance:
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“It is not difficult to make
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microbes resistant to penicillin
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in the laboratory by exposing
them to concentrations not
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sufficient to kill them, and the
Fleming (centre) receiving the Nobel prize same thing has occasionally
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APPL. MICROBIOL. 1963; 11: 122-127
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JAMA. 1964;189:829-834
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TEM-1
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The first plasmid-mediated – β
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lactamase in gram-negatives
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(TEM-1), was described in 1965
from Dr Polixeni Kontomichalou in
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a single strain of E. coli isolated
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during 1964 from a blood culture of
a patient named Temoniera in
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Greece, hence the designation
TEM.
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TEM-2
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Isoelectric
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point
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TEM-1 pI 5.4
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1965
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Gln39→Lys
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TEM-2 pI 5.6
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1970
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Identical biochemical properties
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R- TEM-1+ CTX-1
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Amoxicilin 2 1024 >2048
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Piperacillin 1 128 256
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Piperacillin/ Tazobactam 4mg/L 0.5 1 2
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Cefotaxime 0.03 0.03 8
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Ceftazidime 0.25 0.5 16
Aztreonam
n 0.06 0.12 8
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Cefoxitin 4 4 8
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enzyme, since an intragenic
probe of the TEM-1 gene
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hybridized with a fragment of
the plasmid carrying CTX-1
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Fragment patterns of plasmids harbouring CTX-1.
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Sources of plasmids in E. coli transconjugants were as follows.
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Lane 1: E. coli
Lane 2: K. pneumoniae
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Lane 3: C. freundii
Lane 4: E.aerogenes
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Lane 5: P. morganii
Lane 6: S. marcescens
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Activity vs
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3rd gen cephs
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TEM-1
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1965
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Gln39→Lys
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TEM-2
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1970
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Gln39→Lys Glu104→Lys Gly238→Ser
CTX-1/
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TEM-3
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1987
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Extended-spectrum β-lactamases
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Definition:
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ESBLs are β-lactamases of functional group 2be
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or molecular class A that:
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Hydrolyze:
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¾Third generation cephalosporins
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¾Monobactams
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•at a rate that is equal to or higher than 10% of
n that for benzylpenicillin
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Do not appreciably hydrolyse cephamycins
(cefoxitin or cefotetan) or carbapenems
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R- TEM-1+ TEM-3+ TEM-10+
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Amoxicilin 2 1024 >2048 1024
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Piperacillin 1 128 256 >128
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Piperacillin/ 0.5 1 2 2
Tazobactam 4mg/L
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Cefotaxime 0.03 0.03 8 0.25
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Ceftazidime 0.25 0.5 16 128
Aztreonam
n0.06 0.12 8 64
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Cefoxitin 4 4 8 4
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β-lactamases
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Serine Enzymes Metallo-Enzymes
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Active site
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Mechanism of action (Serine based)
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f β-Lactamases catalyse the hydrolytic cleavage of the β-lactam ring of
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penicillins, cephalosporins and monobactams
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f The products of fragmentation lack antibiotic activity.
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Active
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enzyme f The serine residue irreversibly reacts
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with the carbonyl carbon of the
β-lactamase n lactam ring, resulting in an open ring
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Inactive
penicilloate (inactive lactam) and regenerating
the lactamase.
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cephalosporins
monobactams
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Mechanism of action (Metallo –β- lactamases)
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f A divalent transition metal ion,
most often zinc, linked to a
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histidine or cysteine residue or
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both, reacts with the carbonyl
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group of the amide bond.
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f React with penicillins
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cephalosporins
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carbapenems
not monobactams
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Synthesis and mode of transfer
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The synthesis of lactamases is:
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chromosomal (constitutive)
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Pseudomonas aeruginosa
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plasmid mediated (inducible)
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Aeromonas hydrophila
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Staphylococcus aureus.
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The genetic environment of the β-lactamase
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(bla) gene dictates whether the β-lactamases
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are produced in a constitutive or inducible manner.
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transferred between
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β-lactamase classification
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fMolecular classification scheme
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f The simplest classification scheme
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f Based upon amino acid sequence similarity
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f β-lactamases can be divided into four evolutionary distinct
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molecular classes (A, B, C and D), each with distinct
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sequence motifs
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fFunctional classification scheme
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f According to the substrate profile, inhibitor profiles, and
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Philos Trans R Soc Lond B Biol Sci 1980; 289:321-31
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Class A: serine enzymes
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¾ Staphylococcus aureus PC1 penicillinase
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¾ All previously reported serine β-lactamases with extensive sequence homologies with
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each other and with a preference for penicillin substrates are grouped as class A
enzymes.
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Class B: Zinc-requiring enzymes
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¾ Bacillus cereus β-lactamase II
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Class C: AmpC enzymes from gram-negative bacteria
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¾ The chromosomally encoded β-lactamases of many species of Enterobacteriaceae have
extensive sequence homologies. They therefore constitute a third group of β-
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lactamases (class C) that are serine enzymes but probably have an evolutionary origin
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¾ Hydrolysis rates for cloxacillin and oxacillin higher than that for benzylpenicillin
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¾ Were placed in a separate molecular class from the other serine β-lactamases at 1987
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PC1 β-lactamase of Staphylococcus aureus
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TEM, SHV, CΤΧ-Μ plasmid-β-lactamases
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Inducible cephalosporinases from Klebsiella, P.vulgaris, B.fragilis
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B Metallo-β-lactamases
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AmpC enzymes from gram-negative bacteria; MIR-1
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D OXA-type enzymes
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Ambler Molecular classification system
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β-lactamases
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Active site Serine Enzymes Metallo-Enzymes
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Spectrum of antimicrobial substrate profile
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Enzyme inhibition profile
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Enzyme net charge (pI)
Hydrolysis rate (vmax)
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Binding affinity (km)
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Isoelectric focusingn
Protein molecular weight
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Amino acid composition
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BMJ vol. 327, 22 November 2003
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Functional classification of lactamases
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Year Author Basis of classification of lactamases
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1968 Sawai et al Cephalosporins versus penicillins as substrates
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1973 Richmond and Sykes Expanded substrate profile
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Suggested five major groups (Ia-d, II, III, IV, V)
Differentiate plasmid mediated β-lactamases on
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1976 Sykes and Matthew
the basis of isoelectric focusing
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1981 Mitsuhashi and Inoue Add the category “cefuroxime hydrolysing
nlactamase”
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1989 Bush Expanded the substrate profile
Add the reaction with EDTA
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β-lactamases
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«Bush, Jacoby and Medeiros»
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f Four major groups of enzymes are defined by their substrate and
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inhibitor profiles:
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f group 1: cephalosporinases that are not well inhibited by clavulanic acid;
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f group 2: β-lactamases that are generally inhibited by active site-directed β-
lactamase inhibitors and that belong to molecular classes A or D;
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f group 3: metallo β-lactamases that hydrolyze penicillins, cephalosporins, and
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carbapenems and are poorly inhibited by classical β-lactamase inhibitors except
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EDTA and p-chloromercuribenzoate (pCMB)
f group 4: penicillinases that are not inhibited by clavulanic acid
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Functional characteristics have been correlated with molecular structure
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β-lactamases
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Ambler Bush Hydrolytic activity Inhibited by Representative enzymes
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1960 et al
1995
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Penicillin Cefotaxime Imipenem Clavulanic acid EDTA
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Α 2a +++ - - ++ - Penicillinases from Gram(+) bacteria
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2b +++ - - ++ - ΤΕΜ-1, ΤΕΜ-2, SHV-1
2be +++ ++ - ++ - TEM- και SHV-type, CTX-M-type,
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other ESBL
2br +++ - - - - IRT
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2c ++ - - + - PSE-1, PSE-3, PSE-4
2e ++ ++ - ++ - Inducible cephalosporinases from
n Proteus vulgaris
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2f ++ + ++ + - NMC-1 from E.cloacae, Sme-1 from
S.marcecens
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D 2d ++ - - ± - OXA-1 to OXA-95
B 3 ++ ++ ++ - + L1 from Xanthomonas maltophilia,
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Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular
structure. Antimicrob Agents Chemother 1995;39: 1211—1233.
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Total No of β- Lactamases :
>890
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KPC
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(n=11)
Today: ~500
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TEM: 188 MβL
SHV: 138 (n=68)
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CTX-M: 123
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Today: 132
CMY-type and TEM-1 ESBL
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other AmpC-type Today: 227
β-lactamases 18 ESBL activity
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CTX-M-1 47 Carbapapene-
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mase activity
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Penicillinases
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OXA-1
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from 1970 to 2009
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β-Lactamase Classification and Amino Acid Sequences for TEM,
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SHV and OXA Extended-Spectrum and Inhibitor Resistant Enzymes
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George Jacoby Karen Bush
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Lahey Clinic Indiana University Bloomington
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Burlington, MA 01805 Bloomington, IN 47405
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Phone: (781) 744-2928 Phone: (812) 855-1542
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Fax: (781) 744-5486 n Fax:
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Email: george.a.jacoby@lahey.org Email: karbush@indiana.edu
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Proposed β-lactamase classification by Giske et al, 2009
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Expands the definition of
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ESBL to other clinically
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important acquired β-
lactamases with activity
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against extended-spectrum
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cephalosporins and/or
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carbapenems
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ESBLA ESBLM ESBLCARBA
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Class A ESBLs
Μiscellaneous
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ESBLs
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historical definition: differentiate ESBLs from their parental
enzymes that did not hydrolyse extended-spectrum β-lactams.
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Clinical definition: ESBL-producing organisms are resistant to
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extended-spectrum cephalosporins and monobactams, but can
be treated successfully with carbapenems.
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ESBLCARBA is unnecessary.
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ID
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Expanded classification schemes for bacterial β-lactamases
Bush & Jacoby 2010
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Classification Distinctive Inhibited by Characteristics Representative
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substrate(s)
Bush- Bush- Ambler CA EDTA enzyme(s)
Jacoby Jacoby- (1980) or
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(2010) Medeiros TZB
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(1995)
1 1 C Cephalosporins - - Greater hydrolysis of E. coli AmpC, P99,
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cephalosporins than ACT-1, CMY-2,
benzylpenicillin; FOX-1, MIR-1
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hydrolyzes cephamycins
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b
1e NI C Cephalosporins - - Increased hydrolysis of GC1, CMY-37
ceftazidime and often
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other oxyimino-β-lactams
3a 3 B (B1)
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Carbapenems
n - + Broad-spectrum
hydrolysis including
IMP-1, VIM-1,
CcrA, IND-1
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carbapenems but not
monobactams
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FEZ-1
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Preferential hydrolysis
3b 3 B(B2) Carbapenems - + of carbapenems CphA, Sfh-1
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NI 4 Unknown
a CA, clavulanic acid; TZB, tazobactam.
b NI, not included
Classification Distinctive Inhibited by Characteristics Representative
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Bush- Bush- Ambler substrate(s) CA or EDTA enzyme(s)
a
Jacoby Jacoby- (1980) TZB
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(2010) Medeiros
(1995)
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2a 2a A Penicillins + - Greater hydrolysis of benzylpenicillin PC1
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than cephalosporins
2b 2b A Penicillins, + - Similar hydrolysis of benzylpenicillin TEM-1, TEM-2,
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Cephalosporins and cephalosporins SHV-1
2be 2be A Extended-spectrum + - Increased hydrolysis of oxyimino-- TEM-3, SHV-2,
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cephalosporins, lactams (cefotaxime, ceftazidime, CTX-M-15, PER-1,
monobactams ceftriaxone, cefepime, aztreonam) VEB-1
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2br 2br A Penicillins - - Resistance to clavulanic acid, TEM-30, SHV-10
sulbactam, and tazobactam
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2ber NI A Extended-spectrum - - Increased hydrolysis of oxyimino— TEM-50
cephalosporins, lactams combined with resistance to
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monobactams clavulanic acid, sulbactam, and
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tazobactam
2c 2c A Carbenicillin + - Increased hydrolysis of carbenicillin PSE-1, CARB-3
2ce NI A Carbenicillin,
cefepime
n + - Increased hydrolysis of carbenicillin,
cefepime, and cefpirome
RTG-4 (CARB-10)
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2d 2d D Cloxacillin ± - Increased hydrolysis of cloxacillin or OXA-1, OXA-10
oxacillin
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2, 2a, 2b, 2c, and 2d 2df, 2f, and group
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β-lactamases 3 β-lactamases
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1, 1e, and 2e
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enzymes
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ID
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Extended-spectrum β-lactamases
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f TEM, SHV & CTX-M type
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fAmbler’s class Α
fBush et al group 2be
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AmpC
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f Species-specific AmpC chromosomal enzymes
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f Plasmid-encoded AmpC cephalosporinases
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fAmbler’s class C
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fBush et al group 1
Carbapenemases
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f Serine carbapenemases
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ID
Metallo-β-lactamases
fAmbler’s class Α, Β και D
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Enzyme Molecular Functional group or No of enzymes Representative enzymes
Family class subgroup
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TEM A 2b, 2be, 2br, 2ber 183
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A 2b 14 TEM-1, TEM-2, TEM-13
A 2be 82 TEM-3, TEM-10, TEM-26
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A 2br 36 TEM-30 (IRT-2), TEM-31 (IRT-1), TEM-163
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A 2ber 11 TEM-50 (CMT-1), TEM-158 (CMT-9)
SHV A 2b, 2be, 2br 137
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A 2b 31 SHV-1, SHV-11, SHV-89
A 2be 45 SHV-2, SHV-3, SHV-115
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A 2br 6 SHV-10, SHV-72
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CTX-M A 2be 121 CTX-M-1 to CTX-M-123
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PER A 2be 7 PER-1 to PER-7
VEB A 2be n 7 VEB-1 to VEB-7
GES A 2f 16 GES-2 to GES-17
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KPC A 2f 10 KPC-2 to KPC-11
SME A 2f 3 SME-1, SME-2, SME-3
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– Bush group 2be
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TEM-, SHV- family
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Arose by amino acid substitutions in TEM-1, -2 (82 enzymes) or SHV-1 (45
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enzymes).
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First recognized in 1983. Their emergence and rapid dissemination have
been responsible for numerous outbreaks of infection throughout the world.
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Mostly found in K. pneumoniae and E. coli (also in C. freundii, Enterobacter
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aerogenes, and Serratia marcescens).
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Mostly encoded by large plasmids (up to 100 kb and even more) that are
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transferable from strain to strain and between bacterial species.
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Hydrolyse penicillins, cephalosporins (except cefoxitin), monobactams.
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– Bush group 2be
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CTX-M-1 έως CTX-M-123
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Derivatives of chromosomal genes resident in members of the genus
Kluyvera.
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Clustered in six groups (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, CTX-M-25,
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CTX-M-45) with similarity at the amino-acid sequence level.
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The first CTX-M-type enzyme of clinical origin, CTX-M-1, was described in
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enterobacterial strains isolated in Europe in the late 1980s
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Within a decade, the CTX-M β-lactamases became the predominant ESBL
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family in many medical centres such that they have largely replaced most of
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the TEM- and SHV-derived ESBLs throughout the world.
Most (but not all) CTX-M enzymes hydrolyze cefotaxime more readily than ceftazidime. Many
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f Class C cephalosporinases, Group 1 β-lactamases
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f Are encoded primarily in the chromosome of many species of the
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Enterobacteriaceae
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f Hydrolytic activity
f 1st gen cephalosporins –rapid
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f 2nd and 3rd gen cephalosporins –slow but kinetically efficient
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f 4th gen cephalosporins –slow, kinetically inefficient
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f Carbapenems –nearly stable
Basal in:
n Inducible in:
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E. coli & shigellae Enterobacter spp.
C. freundii
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M. morganii
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Serratia spp.
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P. aeruginosa
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plasmid-mediated AmpC β-lactamases
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f Increasingly reported worldwide (since 1989)
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f Escaped from the chromosome of natural AmpC-producing
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species of the Enterobacteriaceae
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f Are categorized into six groups
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Group Origin Homology Members
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CMY-2 Citrobacter freundii 96% 85
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MIR-1 & ACT-1 n Enterobacter spp 98-99% 15
DHA Morganella morganii 99% 8
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ACC-1 Hafnia alvei 99% 4
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Cephalosporins MIC (mg/L) Disk (µg) Diameter (mm)
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S≤ R> S≥ R<
Cefadroxil 16 16 30 12 12
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Cefuroxime 8 8 30 18 18
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Cefotaxime 1 2 5 21 18
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Ceftriaxone 1 2 30 23 20
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Ceftazidime 1 4 10 22 19
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Aztreonam 1 4 30 27 24
Cefepime 1
n 4 30 24 21
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Cefoxitin NA NA 19 19
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Cephalosporins MIC (mg/L) Disk (µg) Diameter (mm)
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S≤ R> S≥ R<
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Cefazolin 1 2 NA NA
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Cefuroxime 8 16 30 18 15
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Cefotaxime 1 2 30 26 23
Ceftriaxone 1 2 30 23 20
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Ceftazidime 4 8 30 21 18
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Aztreonam 4 8 30 21 18
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Cefepime 8
n 16 30 18 15
Cefoxitin 8 16 30 18 15
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Eliminate the need to perform ESBL screen and confirmatory tests for
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Detection and confirmation are less accurate when multiple enzymes are
present.
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mechanisms.
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CONFIRMATORY TESTS FOR ESBLS
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Double disc tests.
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Cefotaxime, ceftazidime, cefepime and aztreonam disks 30 μg are
applied 25-30 mm away from a co-amoxiclav 20+10 μg disk.
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When the zone of either cephalosporin is expanded by the clavulanate ÆESBL
production
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cheap
critical disc spacing Ævaries with the strain, some producers may
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be missed.
Combination disc methods.
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Cefotaxime or Ceftazidime 30μg disks
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Cefotaxime or Ceftazidime plus clavulanate 30+10μg disks
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Zone diameters of ≥5 mm in the presence of the clavulanate Æ ESBL
production
cheap
n
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do not require critical disc spacing
Etest ESBL strips.
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μg/ml
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f Induction by FOX f Inhibition by 3-aminophenylboronic
(Reduction in CAZ zone diameter) acid or cloxacillin
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FOX
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f Cover leaf test
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n f Εtest Cefotetan /
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Cefotetan+Cloxacillin
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ES
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ID
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And now its time for Carbapenemases
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ID