beta lactamases, ESBL, nitrocepin, penicillin, MIC

1. Beta Lactamases
&
Extended Spectrum Beta Lactamases
Dr.M.Malathi

2. 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

3. 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.

4. Beta lactam antibiotics

7. 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

8. Mechanism of action

9. 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

10. 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.

11. Types
• Inducible – turned off without drug – plasmid
mediated - Eg: Staph aureus
• Constitutive – SHV – 1 chromosomal enzyme
of Kleb pneumoniae - responsible for
ampicillin resistance

12. Mechanism of resistance

13. Regulation of resistance

14. Organisms tested for beta lactamase
• Staphylococcus aureus
• CONS
• Enterococci sp.,
• Neisseria gonorrhea
• Hemophilus influenzae
• Moraxella catarrrhalis

15. Methods of detection
Phenotypic method:
• Acidometric method
• Iodometric method
• Nitrocefin method
• penicillin disc diffusion test
• Penicillin broth microdilution test
• Penicillin zone edge test
Genotypic test:
• PCR for blaZ gene

16. 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

17. 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

18. 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

19. 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.

20. Nitrocefin disk test

22. 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

23. 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

24. Penicillin zone interpretation

25. 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.

26. Interpretation
• A positive beta lactamase production means that
the test organism is resistant to following
antibiotics:
1. Penicillin
2. Amoxycillin
3. Ampicillin
4. Piperacillin
5. Mezlocillin
6. Carbenicillin

27. TREATMENT
• Beta lactamase resistant semisynthetic penicillin(
methicillin, nafcillin, cloxacillin,dicloxacillin)
• Beta lactam – betalactamase inhibitor combinations (
eg: amoxicillin clavulunate, Ampicillin sulbactam,
Piperacillin tazobactam)

28. Extended Spectrum Beta Lactamases

29. 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

30. Epidemiology

32. Diversity of ESBL types
• SHV
• TEM
• CTX – M
• Toho beta lactamases
• PER

33. 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

34. 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

35. • 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.

36. 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.

37. 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

38. PER
• 25% similarity to TEM and SHV type of ESBLs.
• First detected in Pseudomonas aeruginosa and
later in Salmonella Typhimurium and
Acinetobacter isolates.

39. ESBL producing organisms
• Escherichia coli
• Klebsiella sp.,
• Enterobacter sp.,
• Proteus sp.,
• Salmonella sp.,

40. • 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

41. Risk for ESBL infection
• Seriously ill patients
• Prolonged hospital stay
• Invasive medical devices
• Cross infections
• Colonizers in medical staffs
• Immunocompromised
• Prolonged antibiotic intake

42. 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)

43. ESBL detection
• Phenotyping
• Genotyping
Why 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

44. PHENOTYPIC METHODS
( CLSI M100 – S24)
• Screening test:
Disk diffusion test
• Confirmatory test:
Double disk diffusion test
Broth microdilution test

45. Disk diffusion screening test
• For Escherichia coli and Klebsiella sp.,:
1. Cefpodoxime (10µg) ≤17mm
2. Cefotaxime (30µg) ≤27mm
3. Ceftriaxone (30µg) ≤25mm
4. Ceftazidime (30µg) ≤22mm
5. Aztreonam (30µg) ≤27mm

46. • For Proteus mirabilis:
1. Cefpodoxime (10µg) ≤22mm
2. Ceftazidime (30µg) ≤22mm
3. Cefotaxime (30µg) ≤27mm
Use of more than one antimicrobial agent for
screening improves the sensitivity of ESBL
detection.

47. 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

48. ≥ 5 mm disk zone difference

49. 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

50. ≥ 3 twofold concentration decrease in
MIC

51. 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.

52. Interpretation
• For all confirmed ESBL producing strains
• Report as resistant to all penicillins,
cephalosporins and aztreonam

53. 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

54. GENOTYPIC METHODS
• Pulsed field gel electrophoresis
• Polymerase chain reaction
• Ribotyping
• Plasmid profile analysis
• Ligase chain reaction

55. 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

56. 4. Institute contact isolation precautions,
particularly if clonal spread is demonstrated.
5. Institute controls on antibiotic use,
particularly if numerous strain types are
demonstrated.

57. 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

58. 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.

59. 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)

60. 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