Detection of Extended Spectrum Beta Lactamase Producing Strains among Clinical Isolates of Escherichia Coli and Klebsiella Pneumoniae in Alexandria Using Chrom-ID ESBL Agar and Molecular Techniques

Abstract

Background: Antibiotic resistance has become a serious global problem and affects almost every bacterial species for which treatment with antibiotics is available. The increased prevalence of Enterobacteriaceae producing ESBLs creates a great need for identifying these organisms for infection control and epidemiological surveillance. Objectives: In this study, the aim was to (i) evaluate different phenotypic methods for detection of ESBL producing E. coli and K. pneumoniae (ii) Evaluate the diagnostic performance of the selective chromogenic culture media, chromID ESBL, (iii) Determine the prevalence of the genes blaTEM, blaSHV, and blaCTX-M, which are responsible for ESBL production in clinical isolates of E. coli and K. pneumoniae. (iv)Identify the sequence types of the most prevalent ESBL genes among E. coli and K. pneumoniae in Alexandria. Methodology: One hundred isolates of ESBL producing E. coli and K. pneumoniae isolates were collected. Phenotypic characterization of ESBLs by combined disc test, double disc synergy test (DDST), and the modified double disc synergy test (MDDST) was performed simultaneously with antibiotic susceptibility testing. ChromID ESBL agar (bioMerieux) was evaluated for ESBL detection. ESBLs positive strains were tested for the presence of ESBL encoding genes by PCR. After amplification, twelve TEM, SHV and CTX-M genes were subjected to nucleic acid sequencing. Results: An ESBL phenotype was confirmed in 100/173 (57.8%) isolates by combined disc test. The sensitivity of DDST was 44% when the distance kept at 20 mm. Cefepime yielded the highest performance among the β-lactams, with sensitivity 70% when tested with amoxicillin-clavulanate and 86% when tested with piperacillin-tazobactam. The highest sensitivity among phenotypic methods (98%) was obtained with chromID ESBL. PCR of TEM, SHV and CTX-M genes revealed that 80% were positive for at least one of the studied genes. Sequencing of the randomly selected genes revealed that the eight bla TEM amplification products were TEM-1, while the two bla SHV were SHV-11 and the two bla CTX-M genes were CTX-M-15. Conclusion: using only one disk combination might fail to detect ESBL production. The MDDST using cefepime and piperacillin-tazobactam gives the highest sensitivity followed by MDDST using cefepime and amoxicillin-clavulanate. Agars with chromogenic substrates could be applicable as screening methods for ESBLs producing strains.