Phenotypic Detection Of Extended Spectrum Beta Lactamases Producing Organism Among Godfrey Okoye University Students

FIG 1: CAZ……………………AMC……………………CTX

Phenotypic confirmation of ESBL production using DDST(Afunwa, 2018).

The image in FIG 1 shows an increased zones of inhibition indicating the presence of ESBL producing organism.

1.6.2 ESBL Etests

ESBL Etests have been developed in order to quantify the synergy between extended-spectrum cephalosporins and clavulanate. The Etests called CT/CTL, TZ/TZL and PM/PML are two-sided strips containing gradients of cefotaxime (CT), or ceftazidime (TZ) or cefepime (PM), either alone (at one end of the strip), or combined with clavulanate 4 mg/L (on the other end). The ESBL test is considered as positive when the MIC value of the tested drug is reduced by more than three doubling dilution steps (MIC ratio ≥8) in the presence of clavulanate. The test is also considered as positive when there is either: (a) a rounded zone (phantom zone) just below the lowest concentration of CTL, TZL or PML gradients, or (b) a deformation of the CT, TZ or PM inhibition ellipse at the tapering end. The presence of a phantom zone or an ellipse deformation indicates ESBL production. Interpreting results of the ESBL Etest strips is delicate and requires training(Brossieuret al, 2008).

1.6.3 COMBINATION DISK METHOD

Several manufacturers have developed ESBL detection tests based on the combination disk method. The principle of this method is to measure the inhibition zone around a disk of cephalosporin and around a disk of the same cephalosporin plus clavulanate. Depending on the disk type, a difference of ≥5 mm between the two diameters (i.e., corresponding to a two-fold dilution), or a zone expansion of 50% are considered as indicating ESBL production. The test is easy to perform and its interpretation is straightforward. Sensitivity and specificity for this method were first reported to be 96% and 100%, respectively. Evaluation of the performance of the Oxoidcefpodoxime 10 ng ± 1 μgclavulanate combination disks to distinguish ESBL producers from AmpC overproducers and Klebsiellaoxytoca isolates overexpressing K1 enzyme was done. The presence of clavulanate enlarged the zone of inhibition by ≥5 mm for all 180 ESBL-producing organisms, and by <1 mm for AmpC overproducers and K. oxytoca isolates overexpressing K1 enzyme (Brossieuret al, 2008).

1.6.4 AUTOMATED METHOD

The VITEK 2 ESBL test is based on the simultaneous assessment of the antibacterial activity of cefepime, cefotaxime and ceftazidime, measured either alone or in the presence of clavulanate. This test relies on card wells containing 1.0 mg/L of cefepime, or 0.5 mg/L of cefotaxime or ceftazidime, either alone or associated with 10 or 4 mg/L of clavulanate, respectively. After inoculation, cards are introduced into the VITEK 2 machine, and for each antibiotic tested, turbidity is measured at regular intervals. The proportional reduction of growth in wells containing a cephalosporin combined with clavulanate is then compared with that achieved by the cephalosporin alone and is interpreted as ESBL-positive or – negative through a computerized expert system (Advanced Expert System). (Brossieuret al, 2008). The automated Phoenix ESBL test (Becton Dickinson, Sparks, MD, USA) also relies on the growth response to selected expanded-spectrum cephalosporins. This test is composed of five wells, each containing a cephalosporin alone or in combination with clavulanic acid (cefpodoxime, ceftazidime, and ceftazidime with clavulanic acid, cefotaxime with clavulanic acid and ceftriaxone with clavulanic acid). In this system, the results are also interpreted through a computerized system. (Brossieuret al, 2008)