First Sequence Analysis of Genes Mediating Extended-Spectrum Beta-Lactamase (ESBL) bla-TEM, SHV- and CTX-M Production in Isolates of Enterobacteriaceae in Southern Benin

Abstract

: The production of extended-spectrum beta-lactamase (ESBL) by Enterobacteriaceae is a global public health problem. The present study was carried out on 156 strains of enteric bacteria isolated from urinary and cervicovaginal fluid samples. Identification of the strains was performed using MALDI-TOF MS and antibiotic susceptibility tests by disk diffusion method on Mueller Hinton agar in accordance with the recommendations of the Antibiogram Committee of the French Society for Microbiology. ESBL genes were sought by real time-polymerase chain reaction (RT-PCR) and by gel-based PCR. Gel-based PCR products were used for sequencing of the resistance genes, which were analyzed in the NCBI and Arg Annot databases. Results showed a predominance of Escherichia coli both in the urinary and cervicovaginal fluid samples. Klebsiella pneumoniae was the second most isolated bacterium in the specimens. Sensitivity to antibiotics revealed high levels of cephalosporin resistance but low resistance to carbapenem. No resistance was noted to colistine. The bla-TEM gene was present in Escherichia coli, while bla-SHV was found in Klebsiella pneumoniae and bla-CTX-M was recovered in both strains. Analysis of the sequences revealed that bla-Tem1 was predominant in bla-TEM and bla-CTX-M-15 was most represented by bla-CTX-M. This study confirms the presence of ESBL-producing Enterobacteria in Benin. This was an epidemiological study aimed at detecting cephalosporin resistance in gram-negative Bacillus isolated from urinary tract and genital infections developed by women. Since the advent of molecular biology techniques for the identification of resistance in bacteria including determination of ESBL resistance genes (i.e., TEM, SHV, CTX-M), no study has been conducted to identify the different variants that circulate in Benin by sequencing these resistance genes. This sequencing is essential in order to differentiate the non-ESBL parental enzymes, which is not possible with the commonly used PCR techniques that do not permit differentiation of the point generating different variants of the ESBL genes. The present study then helped to identify those variants, in particular Tem1, SHV1, and CTX-M15, which are most encountered in Benin and around the world.