RAPID DETECTION OF EXTENDED SPECTRUM Β – LACTAMASES ( ESBL ) AND THEIR CTX-M GENETIC CHARACTERIZATION

Manar Effat Zaiton 1 , Samah Saad EL Din 1 , Nahla Abd EL Hamid 2 , Dalia Salem 1 and Somia EL Sheikh 2 . 1. Microbiology Department, Theodor Bilharz Research Institute (TBRI), Giza, Egypt. 2. Clinical Microbiology and Immunology Department Faculty of Medicine, Zagazig University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

62 using 2 ml of concentrated (pH 8) phenol solution to which 16.6 ml of distilled water was added. Ten microliters of a concentrated tazobactam solution (Sigma-Aldrich, France) (40 mg/ml) was added to tube C. Next, 100 μl of the solution containing pH indicator (phenol red) was added to tube A (Control), and 100 μl of the same solution supplemented with cefotaxime (6 mg/ml) was added to tubes B and C. The three tubes were incubated at 37°C for 20 min. The results were considered negative when all tubes were red and thus interpreted as containing non-ESBL strains. When tube B was yellow/orange and both tubes A and C were red, the test result was considered positive (ESBL-producing isolate). When tube A turned to yellow/red, the test result was considered non interpretable, regardless of any color change for tubes B and C (Nordmann et al., 2012).
Genotypic identification of ESBLs:-DNA Extraction:-All phenotypically confirmed ESBL positive isolates by DDST were analyzed for bla CTX-M. Extraction of DNA from a single colony of cultured bacteria was done by Thermo Scientific Gene JET Genomic DNA purification kit; K 0721(Thermo Fisher Scientific, USA).

PCR Assay:-
The PCR reaction was performed in a final volume of 50 μL with a 5 μL of DNA template, 0.5 μM of each of the forward and reverse primers, 25 μL of master mix (DreamTaq Green PCR Master Mix, Thermo Scientific, USA) and the volume was completed to 50 μL by using 19 μL of sterile nuclease free water. Primers used were consensus designed to catch all groups of CTX-M gene and were described by Pagani et al., 2003 (Table 1). Amplification reactions were carried out in Biometra Germany thermal cycler, under the following conditions: initial denaturation at 95ºC for 3 min, followed by 34 cycle of denaturation at 95ºC for 30 sec, annealing at 49ºC for 40 sec and elongation at 72ºC for 40 sec. The final elongation step was extended to 10 min at 72ºC. The amplified products of the PCR reactions were analyzed by electrophoresis in 2% agarose gel with ethidium bromide, and 100 bp ladder used as DNA molecular weight standard. In each PCR assay, a negative control (lacking DNA) was included.
The sensitivity and specificity of ESBL-NDP test were 97.2% and 100%, respectively in comparison with the DDST whereas the positive and negative predictive values of NDP test were 100% and 97.4%, respectively, with accuracy 98.6%. Kappa testing showed an almost perfect agreement between the ESBL-NDP test and DDST in detecting ESBLs (K # 0.973 = very good agreement).

Statistical analysis:-
Results are expressed as number (%). P value: 0.05 was considered significant and < 0.01 was considered highly significant. Agreement between the different diagnostic tests was tested using kappa statistic. Data analysis was done using computer programs SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 19 windows.

Discussion:-
ESBL production is among the commonest antibiotic resistance problem that has been accelerating worldwide (Oli et al., 2017). The frequent use of β-lactamases has given rise to continuous spread of resistant isolates due to their selective force driving alteration of the resistance mechanisms. Egypt has been recognized as one of the countries with extremely high rate of ESBL producers, with up to 70% of isolates producing the enzyme (Borg et al., 2006). Possibly, this high prevalence is related to the less controlled use of antibiotics in Egypt, where many drugs are still available over the counter (Taha et al., 2015).Therefore, establishing a new and rapid approach in diagnosis of ESBL is crucial in controlling resistance spread. In addition, data concerning dispersion and clonality of CTX-M producing isolates, and molecular epidemiology, arouse attention about rapid spread of CTX-M resistant genes and the high rate of horizontal transfer between different bacterial species (Cantón et al., 2012).
Therefore, we aimed to detect the burden of ESBLs by the rapid ESBL-NDP test, and to examine the prevalence of CTX-M gene among Enterobacteracea in Egypt.
In the present study, ESBL production among Enterobacteriaceae clinical isolates was found to be 49 % by the DDST, which is nearly identical to 48.9% that was detected by Abdallah and colleagues (

Conclusion:-
In conclusion, there is high prevalence of ESBL producing strains in our hospital. The ESBL-NDP test leads the DDST in its rapid ability for detection of ESBL production from bacterial cultures. It is also easy to prepare with cheap in-hand kits, making it a better alternative for ESBL detection in small laboratories. A positive result in the ESBL-NDP test may indicate exclusion of expanded-spectrum cephalosporins for treating such patients. In addition, our study clearly demonstrates the striking change in gene pool of Enterobacteriaceae, as higher rates of bla CTX-M detection confirms mobilization of these genes via plasmids replacing bla SHV and bla TEM resistance genes.