I trust that it has been a problem with phenotypic test to confirm co-producers of AmpC beta-lactamases and ESBL. Is the PCR for AmpC's and PCR-SEQUENCING for ESBLs the only option?
Dear Murugan thank you for your interest in the topic.
Its true that ESBL producers are susceptible to cephamycin (cefoxitin OR cefotetan).
But a when the isolate is resistant to all 3rd generation cephalosporins (such as Ca, Ci and Ce) and also cephamycin (cefoxitin OR cefotetan)- its clear that its a AmpC beta-lactamse producer. But there is no clue that it is a co-producer of AmpC and ESBL!!
If a bacterial strain resistant to cefotetan drug, its AmpC producer.
If a strain resistant to either ceftazidime or cefotaxime will be ESBL Producer.
ESBL producer will not be resistant against Cefotetan group of drug.
But if ESBL co-exist with AmpC, they will be resistant to CEFOTETAN( Cephamycin drug) as well as Ceftazidime and or cefotaxime (Cephalosporin drugs).
By comparing the basic Antibiogram reading itself we could calculate the type of resistance sir.
regards
Murugan N
Dear Murugan thank you for your interest in the topic.
Its true that ESBL producers are susceptible to cephamycin (cefoxitin OR cefotetan).
But a when the isolate is resistant to all 3rd generation cephalosporins (such as Ca, Ci and Ce) and also cephamycin (cefoxitin OR cefotetan)- its clear that its a AmpC beta-lactamse producer. But there is no clue that it is a co-producer of AmpC and ESBL!!
Dear Dr. Godfred sir,
I too agree with your statement.
Am working on this field, will get you soon the right answer.
Dear Murugan I think I have an answer, but will think over again and will let you know.
Dear Murugan and others currently working researchers on broad spectrum beta-lactamases, do let me know if you have come across more simpler phenotypic tests to confirm co-production of AmpC beta-lactamases and ESBL's.
This test is done on a single culture plate. Requirements are, 1. PHOSPHATE BUFFERED SALINE (PBS) 0.1M,pH 6.0{100ml}; 2. CLAVULANIC ACID STOCK SOLUTION IN PBS; 3. PHENYLBORONIC ACID STOCK SOLUTION IN DMSO.
a. Increase in Zone diameter size by ≥ 5 mm in Cpm/CA {cefepime/clavulanic acid] when compared to Cpm alone indicates ESBL
production.
b. Increase in zone diameter size by ≥ 5 mm of Cx{Cefoxitin} alone when compared to Cx/PBA is indicative of AmpC production
c. Increase in the zone diameter of CAZ/CA/PBA {ceftazidime/clavulanic acid/phenyl boronic acid} by ≥ 3mm when compared
to CAZ/PBA is indicative of coproduction of ESBL and AmpC.
AmpC ß –lactamase can be differentiated from extended spectrum ß –lactamases (ESBL) by their ability to hydrolyze cephamycins and not inhibited by clavulanic acidAlthough many phenotypic tests are available for AmpC detection, there is none validated by the Clinical Laboratory Standard Institute (CLSI). AmpC be inhibited by cloxacillin or boronic acid. you can use boronic acid +- cefoxitin for detection of AmpC producing isolates but if your isolates have KPC or GES enzymes my be shown false-positive results.
Dear Alireza thank you, but how would you detect the coproduction of ESBL and AmpC in your lab.
Hi Godfred,
you can do a CLSI combination disc test (cefotaxim vs. cefotaxim+clavulanate and ceftazidim/ceftazidim+clavulanate) on normal Mueller Hinton Plates and on Mueller Hinton Plates supplemented with 200mg/L cloxacillin (commercially available or you can produce them yourself).
Cloxacillin inhibits AmpC - so if you see a a larger inhibition zones on the cloxacillin-plates, your isolate is an AmpC producer. If the CLSI combination disk is negative/non interpretable on the MH plate but positive (>=5mm increase between the disc with/without clavulanate) on the MH+Cloxa plate, then your isolate produces both AmpC and ESBL.
If you don't have cloxacillin, you can also do the CLSI combination disc test (cefotaxim+/- clavulanate, ceftazidim+/- clavulanate) and add cefepim +/- clavulanate.
If there is no/small inhibition using cefotaxim/ceftazidim+/- clavulanate but a marked one using cefepime and an increase of >=5mm between cefepim and cefepim+clavulanate, you are likely to have an ESBL/AmpC coproducer. This test is sometimes more difficult to interpret than the cloxacillin one.
A third test would be Mast D68C: 4 discs (cefpodoxim=disc A, cefpodoxim +ESBL inhibitor=B, cefpodoxim+AmpC inhibitor=C, cefpodoxim + ESBL+AmpC inhibitor=D).
If you see an increase in D only (see attached picture), your isolate produces both ESBL and AmpC.
I would recommend to use the D68C instead of the D69C, as it provides information on both ESBL and AmpC production.
Best of luck,
Axel
An increase of ≥ 5 mm in zone diameter of CAZ, CTX, FOX and/or CPM tested in combination with BA (CAZ, CTX, FOX–BA and/or CPM –BA) versus CAZ, CTX, FOX and/or CPM alone was considered positive for AmpC β-lactamases
A very simple method by MAST Group. I have tried this and found it good.
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