ESBL strains were routinely identified and we randomly selected 46 strains (20 Klebsiella pneumoniae, 23 Escherichia coli and 3 Enterobacter cloacae) including 31 strains isolated at Sylvanus Olympio Teaching Hospital (CHUSO) and 15 strains at InstitutNational d’Hygiène (INH).
- Sylvanus Olympio Teaching Hospital (CHUSO) is the reference center of the country and is located in Lomé, the capital city of Togo
- Institut National d’Hygiène (INH) is the national public health laboratory of Lomé.
These two centers are the ones that carry out the most acts of medical bacteriology in Lomé.
A total of 15 strains were isolated in 2015 (3 Klebsiella pneumoniae and 12 Escherichia coli) and the others in 2016. Strains came from various samples (urine, blood culture, pus, vaginal specimen).
Susceptibility testing
Bacteria were identified for the most part by conventional technique on the basis of their biochemical characters or by API 20E system. They were tested on various antibiotic discsthrough the method of Kirby Bauer and the interpretations made in accordance with the recommendations of the CA-SFM 2015 (Antibiogram Committee ofFrench Society for Microbiology). The antibiotics tested were: beta-lactams (amoxicillin, amoxicillin + clavulanic acid, ticarcillin, ticarcillin + clavulanic acid, cefoxitin, cefotaxime, cefepime, ceftriaxone, imipenem, ertapenem); monobactams (aztreonam), aminoglycosides (gentamycin, amikacin); quinolones (levofloxacin); sulfadoxine-pyrimethamine; fosfomycin.
They were labeled carriers of ESBL in presence of synergy between amoxicillin+ clavulanic acid discs and aztreonam or between amoxicillin+ clavulanic acid and one of the third generation cephalosporin discs.
Molecular Characterisation of ESBL genes
The DNA was extracted by boiling extraction from an isolated colony suspended in 100μlof water at 95 ° C for 15 minutes followed by a centrifugation step. The blaCTXM and blaSHV genes were identified through gene amplification using well defined primers(SHV-A: 5'-atg-cgt-tat-wtt-cgc-ctg-tgt-3 '; SHV-B : 5’-tta-gcg-ttg-cca-gtg-ctc-g-3’; CTXM-A : 5’-scs-atg-tcg-agy-acc-agt-aa-3’; CTXM-B : 5’- ccg-cra-tat-grt-tgg-ttg-tg3’ ; TEM-1 : 5’-gta-tcc-gct-cat-gag-aca-ata-3’ ; TEM-2 : 5'-tct-aaa-gta-tat-atg-agt-aaa-ctt-ggt-ctg-3')according to the following program: 95 ° C for 3min, 95 ° C for 30s, 55 ° C for 30s, 72 ° C for 60seconds, then 72 ° C for 7 minutes with 50 μl of reaction mixture (25 μl of GreenTaq, 2.5 μl of each primer;18 µl of H2O and 2 μl of DNA extract). The search for blaSHV was carried out only on the strains of K. pneumoniae, that of blaCTXM on all the strains without any exception. The gene amplification of blaTEM was carried out only in case of negativity to the two desired genes. The primers used were (TEM-1: 5'-gta-tcc-gct-cat-gag-aca-ata-3 '; TEM-2: 5'-tct-aaa-gta-tat-atg-agt-aaa- ctt-ggt-ctg-3 '). For all the desired genes, the gene amplification was done over 30 cycles. Positive controls for each gene were used as controls. The products of amplification were revealed by a UV reader after electrophoretic migration on a 1% agarose gel using ethidium bromide as intercalating agent. The migration was performed over 60 minutes at 100V with a 100bp size marker (Promega, USA).
Sequencing
All the positive amplicons were purified by a Quiagen Kit, QIAquick PCR Purification Kit (Roche laboratories) and a sequencing PCR for each primer was performed with a 10µltotal mix (2µlof Big Dye, 3µl of primer 1mM, 3µlof Water,2µ1 of Purified extract) according to the following program: 96 ° C for 3 minutes, 96 ° C, 55 ° C for 15sec, 60 ° C for 4 minutes, 60 ° C for 10min over 25 cycles. The resulting PCR products underwenta membrane filtration using the Edge BIO kit and then through the Hitachi ® 3130 Genetic Analyzer Applied Biosystems Sequencer. The sequences obtained were transferred to a computer for analysis. The sequencesediting was done using 4peaks® software and the sequences were subjected to comparison by blastx (www.ncbi.nim.nih). The comparison results were selected on the basis of 97-100% identity.
Molecular characterization was performed at Bacteriology-Hygiene Laboratory of Bicêtre Hospital in Paris.