u may discuss the issue with Dr. Santi M. Mandal, Assistant Professor , Vidyasagar University, email: [email protected]

Dear Gourisankar

Thnx for answer. i will contact wtih dr.

If you see no inhibition zone with the inhibitor combinations ceftazidime/clavulanate and cefotaxime/clavulanate (and you are sure that the discs are o.k.), you should check for AmpC producers (which are usually resistant to this inhibitors). Also, a carbapenemase producer should be ruled out. AmpC and carbapenemases are lactamases, too, but they are not ESBL (which is the confirmation test for).

Hi Ibrahim

Have you considered using nitrocefin in a colorimetric assay for lactamase activity.

It is very easy.  I could send you a ref if of interest.

Liam

Thanks.

Wish the problem will be solved soon.

Thans for your help. @liam i will be very happy if you can

Hi Ibrahim

The method is described in the attached paper.

Basically, nitrocefin is a yellow coloured anilog of lactam antibiotics.  It is cleaved by lactamases in the same way that drugs are cleaved.  However, upon cleavage the colour changes from yellow to red.  You can monitor reduction in yellow of increase in red over time and from the kinetics curve assess lactamase activity.

Let me know if you have any furhter questions after reading the protocol.

Liam

Hi,

We check the presence of beta-lactamase in milk by adding 4 ppb of benzylpenicillin. Part of the milk we check directly and part of the milk after incubation for some 45 minutes at 30-37°C. We check both samples with a microbial inhibitor test or a rapid antibiotic milk test. If we see a difference (degradation) between both results we know that beta-lactamase is present.

Can you add blank milk to your samples?

Thanks for your answers.

As per AMBLER's classification of Beta lactamases there are 4 groups :

A,B,C,D

A,C,D have serine at their active site and B have zinc and metal

ions at their active site.

usually A(TEM/SHV/CTXM) & D are plasmid mediated

and B &C are chromosomal.

Class A & D undergo mutation to produce ESBLs.

Class A/D: Resistant to Penicillin,Cephalosporins and monobactams but

sensitive to Betalactamase inhibitors , cephamycins and carbapenems.

Class

A/DESBLs : Resistant to Penicillin,Cephalosporins and

monobactams,  cephamycins - may or may not be resistant to Betalactamase

inhibitors - sensitive to carbapenems

Class B are called MBLs (Metallo-

betalactamases) : Resistant to Penicillin, Cephalosporins, Betalactamase

inhibitors , cephamycins and carbapenems but sensitive to monobactams

(aztreonam) only.

Class C are called AmpC betalactamases : Plasmid induced

enzymes : Resistant to Penicillin, cephalosporins 1st,2nd and 3rd

generations, beta lactamase inhibitors, cephamycins but sensitive to 4th

generation cephalosporins and carbapenem 

The ideal indicator cephalosporin is one to which all ESBLs confer

resistance, even when their production is scanty. Choice is predicated by

the following general traits:

All ESBLs : Obvious resistant to cefpodoxime

TEM & SHV ESBL : Obvious resistant to ceftazidime

CTX-M ESBL : Obvious resistant to cefotaxime

ESBL Screening “indicator” drugs :

– Cefpodoxime

– Ceftazidime

– Cefotaxime

– Ceftriaxone

– Aztreonam

The use of more than one

antimicrobial agent increases the sensitivity of detection.

ESBL Confirmatory Tests : Double-disk synergy (DDS) test: 5 mm or more  difference in zone of inhbition between

Ceftazidime & Ceftazidime- clavulanate disc

cefotaxime & Cefotaxime-clavulanate disc

when an ESBL-producing isolate is identified, it should be reported

as clinically resistant to all cephalosporins, penicillins, and aztreonam,

despite the in vitro test results.

The carbapenems (imipenem, meropenem,  ertapenem) are active against ESBL-producing strains, as are  the cephamycins (cefoxitin and cefotetan).

Dear sir:

Colorimetric, acidimetric and iodometric tests of beta-lactamase production are good, rapid indicators of penicillin and ampicillin resistance in Haemophilus, Moraxella and Neisseria spp. These methods can also be applied to Gram-negative aerobic bacilli but are less useful. Accurate identification of beta-lactamases of Enterobacteriaceae demands gene or protein sequencing, but the broad type of enzyme produced by an isolate can often be inferred from antibiotic susceptibility data. Resistance to ceftazidime or cefpodoxime implies extended-spectrum beta-lactamase (ESBL) production in Escherichia coli and Klebsiella spp., especially if susceptibility to cefoxitin is retained. ESBL production can be confirmed with double disc tests or with various commercial kits. Antagonism between cefoxitin and cefotaxime can be used to infer the presence of inducible AmpC enzymes in these species.

With my best regards

One test involves using disks (such as Kirby-Bauer disk diffusion test) that contain cefotaxime and ceftazidime alone and disks containing a combination of clavulanic acid with these antibiotics.