Beta Lactamases &

Extended Spectrum Beta Lactamases

Dr.M.Malathi

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

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.

Beta lactam antibiotics

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

Mechanism of action

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

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.

Types

• Inducible – turned off without drug – plasmid mediated - Eg: Staph aureus

• Constitutive – SHV – 1 chromosomal enzyme of Kleb pneumoniae - responsible for ampicillin resistance

Mechanism of resistance

Regulation of resistance

Organisms tested for beta lactamase

• Staphylococcus aureus• CONS• Enterococci sp.,• Neisseria gonorrhea• Hemophilus influenzae• Moraxella catarrrhalis

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

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

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

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

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.

Nitrocefin disk test

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

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

Penicillin zone interpretation

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.

Interpretation

• A positive beta lactamase production means that the test organism is resistant to following antibiotics:

1. Penicillin2. Amoxycillin3. Ampicillin4. Piperacillin5. Mezlocillin6. Carbenicillin

TREATMENT

• Beta lactamase resistant semisynthetic penicillin( methicillin, nafcillin, cloxacillin,dicloxacillin)

• Beta lactam – betalactamase inhibitor combinations ( eg: amoxicillin clavulunate, Ampicillin sulbactam, Piperacillin tazobactam)

Extended Spectrum Beta Lactamases

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

Epidemiology

Diversity of ESBL types

• SHV• TEM• CTX – M • Toho beta lactamases• PER

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

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

• 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.

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.

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

PER

• 25% similarity to TEM and SHV type of ESBLs.• First detected in Pseudomonas aeruginosa and

later in Salmonella Typhimurium and Acinetobacter isolates.

ESBL producing organisms

• Escherichia coli• Klebsiella sp.,• Enterobacter sp.,• Proteus sp.,• Salmonella sp.,

• 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

Risk for ESBL infection

• Seriously ill patients • Prolonged hospital stay• Invasive medical devices• Cross infections• Colonizers in medical staffs• Immunocompromised• Prolonged antibiotic intake

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)

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

PHENOTYPIC METHODS ( CLSI M100 – S24)

• Screening test: Disk diffusion test

• Confirmatory test:Double disk diffusion testBroth microdilution test

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

• 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.

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

≥ 5 mm disk zone difference

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

≥ 3 twofold concentration decrease in MIC

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.

Interpretation

• For all confirmed ESBL producing strains

• Report as resistant to all penicillins, cephalosporins and aztreonam

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

GENOTYPIC METHODS

• Pulsed field gel electrophoresis• Polymerase chain reaction• Ribotyping• Plasmid profile analysis• Ligase chain reaction

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

4. Institute contact isolation precautions, particularly if clonal spread is demonstrated.

5. Institute controls on antibiotic use, particularly if numerous strain types are demonstrated.

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

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.

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)

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