Molecular Characterization Of Extended Spectrum Betalactamases Genes (Blactxm And Blashv) In Enterobacteria Isolates In Medical Specimens In Lomé (Togo)

Extendum Bêtalactamases genes spread throughout the world. Many informations are known it in and In particularly in we have lack informations although their prevalence are still The aim of this study is to identify the blaSHV and blaCTXM genes on Escherichia coli and Klebsiella pneumoniae strains isolated in two medical bacteriology laboratories in Lomé. An

The strains were isolated from various samples in 2015 and 2016. An amplification of blaTEM was carried out in case of negativity to both genes. A sequencing of the amplicons was carried out then the sequences identified through blastX on the basis of NCBI data.

Conclusion
There is a diversity of blaSHV genes with a dominance of blaCTXM-15. blaTEM remains the gene to search for in case of absence of the two previous genes in ESBL strains in Lomé. 3 Background Beta-lactams are a class of antibiotic molecules widely prescribed worldwide in various bacterial infections. The resistance of bacteria to antibiotics is a public health issue. Indeed, the development by resistant bacteria especially to this class of antibiotics significantly reduces the therapeutic options. These include the production of extendedspectrum beta-lactamases (ESBL); enzymes which inactivate all cephalosporins and spare only cephamycins and carbapenems are quite frightening. They are quite widespread worldwide because most often their production is mediated by plasmids (1) and remains a concern at the hospital because responsible for healthcare associated infections (2). Most often, we find the enzymes of CTX-M and SHV types in enterobacteria. SHV is a constitutive enzyme in Klebsiella pneumoniae which first SHV-1 was described in 1972 and since,several variants have been described (3). CTX-M remains by far the most described in ESBL (1).
Data on these enzymes exist at the Asian and European levels, however, are lower at the African level and rare at the national level. However, prevalence of ESBL producing bacteria are mentioned across the continent (4)(5)(6). In order to have epidemiological data at the national level, we have initiated this work which purpose is to describe the types of CTX-M and SHV carried by clinical strains of enterobacteria in Lomé.  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.

Methods
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.
The gene amplification of bla TEM 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,

Antibiotic susceptibility
The different bacteria were all resistant to amoxicillin. Only a few (2.1%) were susceptible to levofloxacin and sulfamethoxazole-trimetroprime beta-lactamase inhibitors. Cefepime and cefoxitin resistance rates were respectively 95.6% and 19.5%. The resistance to gentamicin was 72% (Figure 1). Some cases of resistance to imipenen (6.5%) and ertapenem (8.7%) were observed.

Extendum Spectrum Beta-lactamase genes
All bacteria produced at least one desired beta-lactamase gene. Indeed 97.9% (n = 45) carried bla CTXM and / or bla SHV . bla CTXM was carried by 33 strains mean 72%; all E. coli strains were positive to bla CTXM . All the strains of K. pneumoniae (n = 20) carried one type of bla SHV and for eight (08) of them, which is 40% it was associated with bla CTXM . One strain of E. cloacae was negative to both genes despite the phenotypic presence of a synergistic image between the molecule of aztreonam and that of amoxicillin + clavulanic acid. For the latter we performed an amplification of the bla TEM gene which was positive (Figure 2). At sequencing, all bla CTXM genes identified were bla CTXM-15 for the 33 positive bla CTXM strains. Different bla SHV were identified. The most found type was bla SHV-11 in a proportion of 45% (n = 9). The other types were bla SHV-1 (15%; n = 3), bla SHV -28 (20%; n = 4), bla SHV-61 (10%; n = 2), bla SHV-77 (10%; n = 2). On the CTXM-15 combinations, we found 37.5% bla SHV-11 (n = 3), 37.5% bla SHV-28 (n = 3) and 25% bla SHV-1 (n = 2). The strains carrying the bla SHV-1 / bla CTXM-15 combination were from INH and had almost the same susceptibility profile except that one was susceptible to gentamicin. Among the three strains carrying the bla SHV-11 / bla CTXM-15 combination, two were from INH, one of which was resistant to cefoxitin and the strain isolated from CHUSO was resistant to ertapenem. The strains carrying the bla SHV-28 / bla CTXM-15 combination all came from INH and only one was susceptible to cefalotin. All these strains were isolated between July and  Table 1.

Discussion
In this study, different antibiotic susceptibilities were noted. Indeed ESBL confers resistance to all beta-lactams except cefoxitin and carbapenems and their action is inhibited by beta-lactamase inhibitors such as clavulanic acid. We found that our strains show relatively little susceptibility to amoxicillin + clavulanic acid combination and 19.5% of the strains were resistant to cefoxitin, suggesting a combination of mechanisms. Indeed for some (8.5%), we have a resistance to carbapenems which may explain the insensitivity to inhibitors. Resistance to cefoxitin to some extent may be explained by overexpression of the natural cephalosporinase in K. pneumoniae and E. cloacae. In all cases, these 8 strains are multidrug-resistant, as most, 72%, and 98% are resistant to gentamicin, to sulfamethoxazole-trimetoprim and to levofloxacin, respectively. This multi-resistance was also observed in Senegal (5), Nigeria (7) on strains producing an ESBL and even here in Togo since 2009 (6). Extended spectrum beta-lactamases are disseminated throughout the world. So it is not surprising that we constantly find it in our laboratories of medical bacteriology. SHV is a constitutive beta-lactamase in K. pneumoniae. The first to be described is SHV-1 (3) and since then several variants exist that are either ESBL or not (12). All variants described in our study were all ESBL and some associated with CTXM-15 (www.bldb.eu; https://www.card.ca). The identification of different types of SHV shows us a diversity of circulation of K. pneumoniae strains in Lomé. Two types remain dominant: bla SHV-1 and bla SHV-11 identified in total over 60% of the strains. Indeed bla SHV-11 is a variant of bla SHV-In our study, two other types: SHV-1 and SHV-77 were described on strains in both years suggesting a probable expansion of the same strains.
The association of two types of ESBL as described in our study indicates an exogenous acquisition by transfer of genetic elements. Since most ESBL plasmids are known to carry other antibiotic resistance genes, it is easy to understand the multi-resistance of the strains in this study. bla SHV-28 / bla CTXM-15 was described on strains of K. pneumoniae isolated in Copenhagen, Denmark as being an epidemic clone (14). This association was found in 3 strains isolated between July and August 2016 also making us think of this same hypothesis. However this is to be confirmed by genotypic techniques.
Two of the K. pneumoniae strains carried the bla SHV-61 gene, which was first described in 2009 in Portugal. They were isolated in June and July of the same year in pus from different patients. Without further information on patients, we were unable to investigate the notion of recent or past travel in this country.
bla TEM is a beta-lactamase that was already described in isolates of Escherichia coli and K. pneumoniae in our country (15,16). Its presence on an isolate of Enterobacter cloacae remains a first in our country.
The prevalence of carbapenem resistance in this study is low. Indeed, carbapenems are molecules used as a last resort for the treatment of infections due to ESBL or multidrugresistant bacteria. The existence of some carbapenem-resistant bacteria requires a better understanding of the mechanisms underlying this resistance.

Conclusions
During our study, we noticed a multi-resistance of the various strains isolated. The extended spectrum beta-lactamase genes carried by the strains in the two laboratoriesin