Phenotypic detection of extended-spectrum and AmpC beta-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test.
OBJECTIVE
To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases and to make the existing three-dimensional extract test more convenient for use in routine diagnostic laboratories.


METHODS
ESBL and AmpC producing and non-producing isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as identified by the conventional three-dimensional extract test, were used to evaluate the modified procedures. Whole bacterial cells and freeze-thaw preparations, as beta-lactamase sources, were strategically applied to culture plates near ceftazidime and cefoxitin discs on a lawn inoculum of E. coli ATCC 25922. Technical variations of the test included placing the beta-lactamase-containing inoculum into slits, wells and trenches, or onto the surface as spots at varying distances from the discs, and adding clavulanate or cloxacillin to the three-dimensional inoculum to confirm the presence of ESBLs and AmpC beta-lactamases, respectively.


RESULTS
All the methods adopted for ESBL and AmpC detection by using the whole bacterial cells gave positive results. However, the best results were given by the spot-inoculation method. In modifications using the enzymic extracts, the enhanced growth of surface organisms was better appreciated in the designed modifications compared with the conventional methods.


CONCLUSIONS
The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.
Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India. shahidsahar@yahoo.co.in
M
M
Shahid
Abida
A
Malik
Mithlesh
M
Agrawal
Sanjay
S
Singhal
D001426:Bacterial Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001431:Bacteriological Techniques; D002440:Cefoxitin / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D002513:Cephamycins / Q000494:pharmacology; D019818:Clavulanic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D007711:Klebsiella pneumoniae / Q000201:enzymology; D010641:Phenotype; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000032:analysis / Q000378:metabolism*
D001426:Bacterial Proteins; D002511:Cephalosporins; D002513:Cephamycins; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D002440:Cefoxitin; D002442:Ceftazidime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases