1. Introduction
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 [1]. Thereby, the first antimicrobial resistance surveillance data published by the World Health Organization [2] 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 [3]. The production of Extended-Spectrum β-lactamases (ESBLs) 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 were the Temoneira (TEM), Variable sulfhydryl (SHV) and Cefotaximase-Munich (CTX-M) types [4]. The first 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 his name [5]. The SHV-type ESBL was derived by punctual mutations from the original SHV-1 enzyme, which corresponds to a K. pneumoniae chromosomal penicillinase blaSHV gene [6]. Currently, more than 180 SHV ESBLs variants have been described [7]. 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 [8]. CTX-M is the most prevalent ESBLs worldwide [9]. The CTX-M group (for cefotaximase) originally gave to Enterobacteria a higher level of resistance to Cefotaxime, Ceftriaxone, Cefepime and Aztreonam than to Ceftazidime [10]. Some of them have evolved more recently by mutation (ponctual or not) generating a high level of resistance to Ceftazidime such as CTX-M-15, CTX-M-16, CTX-M-19, CTX-M-23 and CTX-M-32 enzymes [11]. Recently, more than 150 variants of CTX-M have been described and classified into 6 phylogenetic groups: the CTX-M-1 group; CTX-M-2 and Toho-1 group; the CTX-M-8 group; the CTX-M-9 group, the CTX-M-25 group and finally the CTX-M-45 group. These new ESBLs were not closely related to TEM or SHV β-lactamases since they only showed 40% homology with these classic ESBLs [12]. Horizontal dissemination of the genes coding for the CTX-M enzymes occurs via conjugative plasmids but also via other genetic elements such as integrons and ISEcp1 insertion sequences [1]. Besides the so-called major ESBLs, there were minor types ESBLs such as TOHO-type, BES-type, Pseudomonas extended Resistance (PER) type, Vietnam extended-spectrum β-lactamase (VEB) type, Guiana extended-spectrum β-lactamase (GES) type, TEM Like Activity (TLA) type, Serratia fonticola (SFO) type which were less studied [13]. TOHO-type is a variant of CTX-M2c [14]. The blaTOHO gene has been described for the first time at Toho University School of Medicine (Japan) in the urine of a one-year-old girl in E. coli TUH12191 [15]. This gene has been notified in the first time in Argentina in Shigella flexneri in the stool of a 33-year-old woman [14]. TOHO-2 ESBLs 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 [16]. The prevalence of the blaTOHO gene in ESBL-producing by Enterobacteria has not been reported in the literature yet. Investigations work on β-lactamases at Burkina Faso scale were relatively recent and have already identified the presence of TEM, SHV and CTX-M genes, which were responsible for bacterial resistance in Enterobacteria [17]. This study was undertaken with the aim of detecting the blaTOHO gene in ESBLs-producing by Escherichia coli and Klebsiella pneumoniae at Saint Camille Hospital of Ouagadougou (Burkina Faso).
2. Methods
2.1. Type of Study
It was a cross-sectional study conducted at Saint Camille Hospital in Ouagadougou (Burkina Faso) from September to November 2018. Samples collected consisted of stool samples, urine samples and vaginal swab samples from hospitalized patients or out-patients. We collected at all 250 samples from 250 patients. This study included samples (stool, urine and urethral swabs) from patients of both sexes who had a stool culture, uroculture or urethral swab culture during the collection period and in whom the bacteria were resistant to Amoxicillin + Clavulanic Acid and at least one 3rd generation cephalosporin or at Aztreonam. We collected samples from all patients who met the selection criteria. Samples were inoculated on common media like Uri Select medium, Hektoen medium and Salmonella Shigella (SS) medium to allow Enterobacteria growth and then incubated for 24 hours at 37˚C. Subsequently, Enterobacteria that grew on the previous media were subcultured on a Mueller-Hinton (MH) medium and then incubated for 24 hours at 37˚C for antimicrobial analysis [18].
2.2. Antimicrobial Essays
The bacterial strains were identified using Analytical Profile Index (API 20 E) Identification method.
Antibiotic susceptibility and resistance test were carried out on Mueller-Hinton (MH) medium with pure colonies of Escherichia coli and Klebsiella pneumoniae according to the recommendations of the Committee of antibiogram of the French Society of Microbiology [19]. The antibiotic discs used were: Amoxicillin + Clavulanic acid (Augmentin), Cefotaxime, Ceftazidime, Ceftriaxone and Aztreonam. All Augmentin resistant Escherichia coli and Klebsiella pneumoniae and at least one third generation cephalosporin were considered to this study as ESBLs producing Escherichia coli and Klebsiella pneumoniae [20].
2.3. Molecular Characterization of ESBLs
2.3.1. Bacterial DNA Extraction
The boiling method was used to extract DNAs from bacteria [21]. The strains were reactivated by culturing on the MH medium for 18 - 24 hours. An isolated colony was taken from the Petri dishes and suspended in 200 µl of distilled water previously aliquoted in labeled Eppendorf tubes, then followed by immersion in a water bath at 100˚C for 15 minutes to release the genetic material. After immersion in a water bath, the suspension was centrifuged at 12,000 rpm for 10 minutes and the supernatant containing DNA released was transferred to a new Eppendorf tube. The concentration of the DNA was then determined using the Nanodrop-type spectrophotometer (BioDrop UV/V is DUO, Holliston, USA).
2.3.2. Molecular Analysis
The reaction medium for PCR was constituted 25 μl volume composed of the Master Mix, the DNA and the primers for blaTOHO. PCR program consisted of an initial denaturation at 95˚C for 5 minutes followed by 30 cycles (Denaturation 95˚C/59 s, Annealing 50˚C/59 s, Elongation 68˚C/59 s) and a final Extension at 68˚C for 5 minutes. We used Gene Amp Thermocycler PCR System 9700.
PCR amplification of blaTOHO gene was carried out with specific primers provided by Applied Biosystems: TOHO-1A 5’-ATG TGC AGT ACC AGT AA-3’ and TOHO-1B 5’-TAG GTC ACC AGA ACC AG-3’ for blaTOHO with 876 pb as molecular weight [22].
2.3.3. Electrophoresis on Agarose Gel
Agarose gel (1%) for electrophoresis was prepared with 1× TBE buffer with addition of 8 µl Ethidium bromide (BET) 0.5 µg/ml which allowed visualization of the bands in the UV light. An electrophoretic migration at 110 millivolts for 30 minutes was performed on the PCR products using a molecular weight marker (1 kb). The fragments were visualized under UV light (Gene Flash) and the images were recorded [23].
2.4. Data Processing
The clinical data was entered in Excel 2013 and then analyzed with the Standard Statistical Package for Social Sciences (SPSS) version 17.0 for Windows and the EPI Info version 6.0 software. All tests of significance were considered statistically significant at p-value < 0.05 [24].
3. Results
We have found at total 16 stools samples, 22 urines samples and 1 vaginal swab samples positive to Escherichia coli and Klebsiella pneumoniae. All samples had shown an antibiotic resistance profile by ESBL production (Figure 1). Among them 15 patients were male and 24 were female, with a sex ratio of 0.63. The ages were ranged from 22 days to 95 years with an average age of 38 years. There were 20 hospitalized patients and 19 out-patients.
Results for the sensitivity/resistance of the 39 bacterial isolates to the different antibiotics tested showed that 26 strains (66.7%) were resistant to Cefotaxime, 28 strains (71.8%) were resistant to Ceftriaxone, 25 strains (64.1%) were resistant to Aztreonam and 20 strains (51.3%) were resistant to Ceftazidime as shown in Table 1. Table 2 showed the frequency of Escherichia Coli and Klebsiella pneumonia involved in bacterial resistance in the study. We notice that all strains were resistant to Amoxicillin + Clavulanic acid (Augmentin®).
Molecular characterization of the ESBLs by PCR revealed that 25 (64.1%) strains isolateed from patients at Saint Camille Hospital of Ouagadougou (HOSCO) carried the blaTOHO gene as shown by the electrophoresis bands (Figure 2).
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Figure 1. Petri dish representing a synergy image characteristic of ESBL producing by Escherichia coli strain.
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Table 1. Resistance profile of different strains to antibiotics used.
Legend: ATM = Aztreonam, CTX = Cefotaxime, CTR = Ceftriaxone, CAZ = Ceftazidime.
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Figure 2. Agarose gel image showing PCR products of blaTOHO genes in identified isolates. Legend: Lane (M) = Molecular Weight Marker (DNA Ladder (1 kb)); Lanes (1 - 9) = Samples; Lane (C−) = negative control; Lanes 3, 5 and 9 = positive to blaTOHO gene.
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Table 2. Distribution of strains involved in bacterial resistance by ESBL production.
4. Discussion
In this report, we mentioned the occurrence of several Escherichia coli and Klebsiella pneumoniae strains carrying the blaTOHO gene. In order to conduct molecular epidemiology study of blaTOHO gene conventional PCR with electrophoresis on agarose gel has been shown to be useful with their sensibility and specificity. In term of predominance of certains strains responsible of antibiotic resistance, it has been found at Laghouat Hospital (Algeria): 43% Escherichia coli and 30% Klebsiella pneumoniae [25]. Other studies; that were done at the Charles De Gaulle Paediatric Teaching Hospital (CHUP/CDG) of Ouagadougou (Burkina Faso), showed 47.22% for Escherichia coli; 15.55% for Klebsiella pneumoniae and 3.33% for Klebsiella oxytoca [26].
The types of ESBLs found in these studies were CTX-M, SHV and TEM. The prevalence of ESBLs produced by Escherichia coli and Klebsiella pneumoniae were also described in South America (45.4% to 51.9%) [27] and Saudi Arabia (55%) [28].
These results confirm that the overall prevalence of ESBLs production by Enterobacteria fluctuates considerably according to the geographical zones, to the countries and to different hospitals. However, the bacterial strains mainly concerned by antibiotic resistance were E. coli and K. pneumoniae with the high level of ESBLs production [25] [27].
The antibiotic susceptibility profile of the 39 strains tested showed resistance to most of β-lactams antibiotics. These levels of antibiotic resistance in the study could be explained by the misuse of antibiotics. It is currently proved that the use of antibiotics, including third-generation cephalosporin for therapeutic purposes is the most important risk factor in the development of bacterial resistance [26]. Other types of resistance mechanisms could explain these levels of antibiotic resistance like the modification of the membrane permeability, the modification of the antibiotic target, the metabolic pathway change or the efflux phenomena [29].
The molecular characterization of the 39 bacterial strains by PCR showed the TOHO type ESBLs in 25 (64.1%). TOHO-1 enzymes have been described for the first time in Japan and were structurally very close to CTX-M and were therefore classified among this group [11] [30]. This type of ESBL (CTX-M) was frequently encountered in hospitals [9]. This could explain the high prevalence of TOHO enzymes in our study. The first detection of TOHO-1 outside Japan was reported in a strain of Shigella flexneri in the stool of a 33-years-old woman in Argentina [14]. 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 [31]. TOHO-1 was an ESBL that has achieved efficient activity not only against penicillins but also against third-generation cephalosporins [32]. TOHO-2 was reported in Tokyo (Japan) in E. coli isolated from the urine of a β-lactam treated patient [16].
This high prevalence of the blaTOHO 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).
The limitations of our study were the small sample size, the scope of the study which is limited to one hospital and the gene sequencing which was not performed. A large sample size with expanding the study to others hospitals for example would allow to find more accurate results. In addition, sequencing of the TOHO gene would confirm our finding.
5. Conclusions
The objective of this work was to detect Escherichia coli and Klebsiella pneumoniae producing TOHO-type ESBL at Saint Camille Hospital of Ouagadougou (Burkina Faso).
This study revealed that alongside the CTX-M, TEM and SHV genes, there were other once rare types of ESBL such as TOHO which was a subgroup of CTX-M found in Escherichia coli and Klebsiella pneumoniae.
This molecular epidemiological study found the blaTOHO gene in 25 bacterial strains (64.1%) carried by patients in hospitals of Burkina Faso and particularly at Saint Camille Hospital of Ouagadougou.