Frequency of Antibiotic Resistance of Escherichia Coli and Klebsiella Pneumoniae by Production of TOHO-type β-lactamases at Saint Camille Hospital, Ouagadougou (Burkina Faso)

Background: Extended-spectrum β-lactamase (ESBL) appeared some years after the introduction in hospital environment of unhydrolysable or extended-spectrum cephalosporins. Several studies have been reported on the blaTEM, blaCTX-M and blaSHV genes in ESBL producing Enterobacteria, however very few studies reported in the literature are related to blaCTX-M subgroup blaTOHO. TOHO enzymes were responsible for healthcare-associated infections in hospitals and in the community. In Burkina Faso, data related to these types of enzymes were scarce. The purpose of this study was to detect TOHO enzymes in Escherichia coli and Klebsiella pneumoniae in order to know the prevalence of infections related to bacterial resistance due to TOHO enzymes at Saint Camille Hospital of Ouagadougou (Burkina Faso). Materials and methods: The study was conducted rstly by microbiological identication of ESBL-producing by Escherichia coli and Klebsiella pneumoniae using API 20 E gallery; secondly the antibiogram was performed by the diffusion method and nally the molecular characterization was made by conventional PCR to search for the bla TOHO gene. The visualization of the specic bands was made using the ultraviolet lamp (Gene Flash) for the photography of the gels. Data were entered and analyzed using Excel 2013 and EPI Info version 6.0 software. A p-value < 0.05 was considered as signicant. Results: We obtained at all 39 strains constituted by 21 (53.8%) Escherichia coli and 18 (46.2%) Klebsiella pneumoniae. Molecular characterization showed the presence of the bla TOHO gene in 25 bacterial strains (64.1 %). Conclusion: It was therefore established in this study the existence of bla TOHO gene at Saint Camille Hospital in Ouagadougou in Burkina Faso. Our study made it possible to know the distribution of the blaTOHO gene in Escherichia coli and Klebsiella pneumoniae.


Background
Antimicrobial resistance became a threat to public health. It constitutes a growing danger to human health in the whole world; but the hospital has always been considered like the most important risk holder (Bradford PA et al. 2001).
Thereby, the rst antimicrobial resistance surveillance data published by the World Health Organization (WHO, 2018) showed high levels of resistance to several serious bacterial infections in both high and low income countries. Antimicrobial resistance is responsible for about 700,000 deaths a year worldwide and has huge implications for the cost of healthcare (Jasovsky et al. 2016). The production of Extended-Spectrum β-lactamase (ESBL) by Enterobacteria is the main mechanism of the antimicrobial resistance.
Several studies have been conducted on the major genes involved in the production of ESBLs. The most common ESBLs are the Temoneira (TEM), Variable sulfhydryl (SHV) and Cefotaximase-Munich (CTX-M) types (Sadeeq et al. 2018).
The rst plasmid TEM-1-type β-lactamase was isolated in 1965 in Greece from a strain of E. coli isolated in a patient named Temoneira hence the name (Zubair et al. 2015). The SHV-types ESBL are derived by punctual mutations from the original SHV-1 enzyme, which corresponds to a K. pneumoniae chromosomal penicillinase bla SHV gene (Brisse and Verhoef 2001;Haeggman et al. 2004). Currently, more than 180 SHV ESBL variants have been described (Liakopoulos et al. 2016). CTX-M ESBLs were initially described in 1986 in Japan, Germany and France in 1989 (CTX-M-1) and have since spread widely around the world (Thomson and Moland 2000). CTX-M is the most prevalent ESBLs worldwide (Paterson et al. 2005).
The bla TOHO gene has been described for the rst time at Toho University School of Medicine (Japan) in the urine of a one-year-old girl in E. coli TUH12191 (Ishii et al. 1995). This gene has been noti ed in the rst time in Argentina in Shigella exneri in the stool of a 33-year-old woman (Andres et al. 2005).
TOHO-2 ESBL have also been described as produced by E. coli TUH1083. It was categorized as an enzyme similar to TOHO-1 group β-lactamase rather than to mutants of TEM or SHV enzymes (Ling et al. 1998

Results
We have found at total 16 stools, 22 urines and 1 vaginal swab samples positive to Escherichia coli and Klebsiella pneumoniae. All samples had shown an antibiotic resistance pro le by ESBL production (Fig. 1 (Fig. 2).

Discussion
In this report, we mentioned the occurrence of several Escherichia and Klebsiella strains carrying the bla TOHO gene (table III). In order to conduct molecular epidemiology study of bla TOHO gene conventional PCR with electrophoresis on agarose gel has been shown to be useful with their sensibility and speci city.
In terms of predominance of certains strains responsibles of antibiotic resistance it has been found at The molecular characterization of the 39 bacterial strains by PCR showed the TOHO type ESBL in 25 (64.1%). TOHO-1 enzymes have been described for the rst time in Japan and were structurally very close to CTX-M and are therefore classi ed among this group (Tetsuya et al. 1997;Bonnet et al. 2004). This type of ESBL (CTX-M) is frequently encountered in hospitals (Paterson et al. 2005). This could explain the high prevalence of TOHO enzymes in our study. The rst detection of TOHO-1 outside Japan was reported in a strain of Shigella exneri in the stool of a 33-years-old woman in Argentina (Andres et al. 2005). This bacterial strain expressed an enzyme belonging to CTXM2c whose DNA sequencing gave TOHO-1.
There were two types of TOHO enzymes (TOHO-1 and TOHO-2) and their precise prevalence has never been reported in an epidemiological study and the truth of the sequence has been questioned because it was so closely related to CTX-M-2 (Hawkey PM et al. 2008). TOHO-1 was an ESBL that has achieved e cient activity not only against penicillins but also against third-generation cephalosporins (Tatsuro et al. 2002). TOHO-2 was reported in Tokyo (Japan) in E. coli isolated from the urine of a β-lactam treated patient (Ling et al. 1998).
This high prevalence of the bla TOHO gene in our study about antibiotic-resisting bacterial strains could also be explained by a spread of this gene in Africa and at Saint Camille Hospital in Ouagadougou, HOSCO (Burkina Faso). Availability of supporting data "Not applicable"

Con ict of Interest
The authors have no con ict of interest to declare.