Molecular Characterization of β-Lactamase Producing Genes and Integrons in Diarrheagenic Escherichia Coli From Diarrheal Children Less Than Five Years of Age in Ouagadougou, Burkina Faso.


 Background

The aim of this study was to determine the resistance of diarrheagenic Escherichia coli strains to β-lactams antibiotics and to perform the molecular characterization of Extended Spectrum β-lactamases (ESBL) and integrons genes.
Methods

This study was carried out from August 2013 to October 2015 and involved 31 DEC strains isolated from diarrheal stools samples collected from children less than five years of age. The identification and characterization of DEC strains was done through the standard biochemical tests those were confirmed using API 20E and Polymerase Chain Reaction (PCR). The determination of antimicrobial resistance was realized by the disk diffusion method then an amplification of the β-lactamase resistance genes and integrons by PCR was done.
Results

Out of the 419 E. coli strains identified, 31 isolates (7.4%) harbored the DEC virulence genes. From these DEC, 21 (67.7%) were ESBL-producing E. coli. Susceptibility to ESBL-producing E. coli showed that the majority of isolates were highly resistant to amoxicillin (77.4%), amoxicillin clavulanic acid (77.4%) and piperacillin (64.5%). The following antibiotic resistance genes and integron were identified from the 31 DEC isolates: blaTEM (6.5%), blaSHV (19.4%), blaOXA (38.7%) blaCTX−M (9.7%), Int1 (58.1%) and Int3 (19.4%). No class 2 integrons (Int2) was characterized.
Conclusions

Because of the high prevalence of multidrug-resistant ESBL organisms found in this study among pediatric patients, there is a need of stringent pediatric infection control measures.


Abstract Background
The aim of this study was to determine the resistance of diarrheagenic Escherichia coli strains to β-lactams antibiotics and to perform the molecular characterization of Extended Spectrum β-lactamases (ESBL) and integrons genes.

Methods
This study was carried out from August 2013 to October 2015 and involved 31 DEC strains isolated from diarrheal stools samples collected from children less than ve years of age. The identi cation and characterization of DEC strains was done through the standard biochemical tests those were con rmed using API 20E and Polymerase Chain Reaction (PCR). The determination of antimicrobial resistance was realized by the disk diffusion method then an ampli cation of the β-lactamase resistance genes and integrons by PCR was done.

Conclusions
Because of the high prevalence of multidrug-resistant ESBL organisms found in this study among pediatric patients, there is a need of stringent pediatric infection control measures.

Background
Antimicrobial resistance (AMR) is one of the most serious global public health threats in this century which is especially urgent regarding antibiotic resistance in bacteria [1], particularly in Enterobacterales [2]. This phenomenon has arisen globally in both nosocomial and community settings as a consequence of widespread antibiotics' consumption [3]. Enterobacterales are a large order of different types of bacteria including Escherichia coli that commonly cause infections both in healthcare settings and in communities [4]. To survive the effects of antibiotics, some Enterobacterales can produce enzymes called extended-spectrum beta-lactamases (ESBLs) that break down and destroy some commonly used antibiotics, including penicillins and cephalosporins, and make these drugs ineffective for treating infections [4]. Over the last decade, many studies have reported the presence of Extended Spectrum β-lactamases (ESBL)-mediated resistance in Gram negative bacteria causing infections in patients [5][6][7][8][9]. Infections that can be caused by ESBL-producing bacteria include urinary tract infection (UTI), diarrhea, skin infections and pneumonia [10]. Possible medications used to treat ESBL infection include carbapenems, which are useful against infections caused by E. coli or Klebsiella pneumoniae bacteria, fosfomycin, beta-lactamase inhibitors, nonbeta-lactam antibiotics and colistin when other medications have failed to stop the ESBL infection [10]. Unfortunately, the excessive use of antibiotics, in particular β-lactams, leads to the selection of ESBL producing strains [11]. Because of the emergence and distribution of Multidrug Resistant (MDR) E. coli is complicating the treatment of various serious infections [12,13], the World Health Organization (WHO) has long recognised the need for an improved and coordinated global effort to contain AMR [1]. The burden of AMR, including MDR, varies between the regions; however, low and middle-income countries share a disproportionate burden due to multitude of factors embedded in the characteristics of the health system, policy, and the practice [14].
In Burkina Faso, there is an emergence of β-lactam resistant enterobacteria, both in rural and urban areas [9,[15][16][17]. Otherwise, carbapenemase-encoding genes are widespread in many parts of the world [18]. According to a previous study, carbapenemase-producing Enterobacterales (CPE) remain one of the most urgent healthcare threats [2].
To this day, the ESBLs and integrons' genes have been poorly characterized in Burkina Faso, particularly in enteric bacteria in children less than ve years of age. However, it is imperative that bacterial isolates from underdeveloped regions undergo extensive MDR characterization to inform national strategies designed to halt the continuing spread of these dangerous pathogens [19]. Therefore, the aim of this study was to determine the resistance of diarrheagenic Escherichia coli strains to β-lactams antibiotics and perform the molecular characterization of Extended Spectrum β-lactamases (ESBL) and integrons genes among clinical DEC isolated from stools collected in children less than ve years of age.

Methods
Study design, area, and sample population It is a cross-sectional study conducted in two hospitals of Ouagadougou, Burkina Faso (Paul VI and Schiphra) during August 2014 to October 2015 (Fig. 1). The Paul VI hospital is located in peripheral area and the Schiphra's hospital in the city center at the dam edge of Ouagadougou. Many patients from Ouagadougou and its surroundings attend these two health care centers because of the good level of health care. The study population comprised children below 5 years attending the hospital for treatment.
The specimens were collected adhering to a standard protocol from pediatric patients below 5 years of age with acute diarrhea and who were hospitalized or visited the health centers as outpatient. Thus, children who attended the hospitals for treatment and provided assent (from parents) or consent for the study were included in the study. Any child over the age of 5 years was excluded from the study.

Sample collection and transport
Three hundred and fteen (315) stool samples were collected in sterile containers and transported to the laboratory of molecular biology, epidemiology and surveillance of bacteria and viruses transmitted by food, centre for research in biological, food and nutritional sciences at the Joseph KI-ZERBO University of Ouagadougou within 24 h in a cool box at + 4°C for immediate analysis.

Screening and con rmation of ESBL and integrons producers
A double synergy test was used for ESBL-producing strains testing. This consisted of placing discs (2-3 cm diameter) of ceftriaxone and cefotaxime around an amoxicillin-clavulanic acid disc on the bacterial plate.
For molecular characterization, DNA extraction was performed using heating method [23]. A loopful of bacterial growth from Mueller-Hinton agar (Lio lchem, Italy) plate was suspended in 1 ml of sterilized water. The mixture was boiled for 10 min at + 100°C and centrifuged for 10 min at 12000 rpm at + 4°C.
Supernatant was then collected and used for the PCR reactions as DNA matrices. Multiplex PCR assays were performed for detecting EBLS-encoding genes (bla TEM , SHV , OXA and bla CTX−M ) and the presence of the class 1, class 2, class 3 integrons from the β-lactams resistant DEC strains. Primers (GeneCust, France) used for these ampli cations are described in Table 1. Antimicrobial susceptibility All the DEC strains tested for the ten β-lactams antibiotics showed important resistances to the aminopenicillins. However, few cephalosporins and carbapenems were yet active on some pathotypes ( Table 2). CTX−M (9.7%) and 2 bla TEM (6.5%). Our results showed that the genes responsible for the production of bla OXA β-lactamases 12/31 (38.7%) were more prevalent in comparison to the genes encoding bla TEM , bla SHV and CTX − M β-lactamases (Table 3). From the three classes of integrons (Int1, Int2 and Int3) assessed among the resistant strains carrying ESBL genes, only 18 Int1 (58.1%) and 2 Int3 (19.4%) was detected. The class 3 integron was detected in only EIEC. No class 2 integrons (Int2) were characterized from the resistant strains. The coexistence of the three resistance genes (bla SHV , bla OXA , bla CTX−M ) and Int1 was found in one EHEC ( Table 3). The bla OXA gene (Fig. 2) was associated with Int1 ( Fig. 3) in 11 cases (p = 0.001) while the bla SHV gene was associated with Int1 in 5 cases (p = 0.100).

Discussion
The emergence and spread of Multidrug Resistant (MDR) bacteria are major public health threats world-wide. Particularly, DEC that produce ESBL are of great concern because their resistance to penicillins and narrow extended-spectrum cephalosporins reduces considerably the treatment options. The prevalence of ESBL in Enterobacteriaceae has been detected at local levels in various african countries; moreover, a study was conducted in 2014 on the prevalence of ESBL and what type of genes are involved in its occurrence [24].  [29], Colombia (11.7%) [30] and Nepal (22.7%) [31]. Otherwise, our result is lower than the ESBL production in clinical isolates of E. coli reported somewhere else in Iran [32]. The prevalence of ESBL resistance in E. coli isolates in European countries is reported to be around 3.9% with variations between countries [33]. Overall, these percentages are lower than those found in middle income countries like Thailand (71.25%) [34] and China (50.5%) [35]. This difference between ESBLs prevalence's might be due to patient's age, the type of samples, the country health facilities in the management of diarrheal infections regarding antibiotics use. Indeed, in developing countries, most patients received antibiotics treatment without prescription [36,37]; such common practices in nearly all developing countries cause a selective pressure on E. coli, whereas in more developed countries effective strategies for the control of antimicrobial are present, which effectively prevents the emergence of ESBLs [36].
It has been reported that bacteria such as E. coli and K. pneumoniae are major ESBL producers resulting in serious threat to the treatment regimen [38]. Indeed, ESBL enzymes are becoming increasingly expressed by many strains of pathogenic bacteria presenting diagnostic challenges to the clinical microbiology laboratories [39,40]. Until recently, antimicrobial therapy has played an important role in the treatment of human bacterial infections. However, the drug resistance has emerged in the treatment of bacterial infections due to ESBL enzymes [39]. Indeed, this enzymes can degrade all β-lactam antibiotics leading to multi drug resistant bacteria. Therefore, reporting of ESBL-producing isolates from clinical samples is critical for the clinicians. It constitutes the guidelines to select appropriate antibiotics for the treatment, including to take proper precaution to prevent the spread of these resistant organisms to other patients [31].
The present study shows 19 ESBLs genes (90.5%) out of the 21 ESBLs producing E. coli. Analysis of the ESBL-encoding genes indicated that the majority of the ESBL-positive isolates harbored bla OXA (38.7%), followed by bla SHV (19.4%), bla CTX−M (9.7%) and TEM (6.5%). The emergence of β-lactam resistance in Enterobacteriaceae is related primarily to the production of enzymes such as TEM and SHV variant which were the most common ESBLs during the past decade. However, OXA and CTX-M β-lactamases have emerged as prevalent ESBL worldwide type compared to the TEM and SHV genotypes [41].
In the present study, OXA-type ESBL producing DEC strains (38.7%) was the most frequently detected ESBL gene. This prevalence is lower than that reported in our previous study in rural area of Burkina Faso: 100% [9], also lower comparatively to 52% reported in Pakistan [42]. However, a recent study in young children reported 3% of commensal E. coli bearing the bla OXA gene in Bangladesh [41]. Thus, it appears that the emergence of ESBLs producing bacteria among gut bacteria of young children can transfer resistance and related genes horizontally across pathogenic E. coli and commensal E. coli leading to a public health concern. Most of the OXA-type ESBL producing E. coli isolates (29%) in our study were detected from the Paul VI hospital (p = 0.002). This hospital is located in peripheral area of Ouagadougou and most of the people living in the slums with poor sanitation conditions attend it for health care sought. Moreover, the provision of confessional care has less di cult accessibility for the peripheral neighborhoods and the population with low socio-economic level. Otherwise, people in Burkina Faso do not consult a health care agent in the case of diseases such as gastrointestinal infections and use self-medication instead [37]. Our results showed 19.4% of SHV-type ESBL producing E. coli which is a little similar to 21% detected in Pakistan [42]. By cons, this prevalence is higher than 0% [9] and 5.9% [17], previously reported in Burkina Faso but lower than 45% reported in Iran [27]. The bla CTX−M gene (Fig. 4) has been detected in three E. coli isolates while its prevalence was 25% in our earlier report [9] and 40.1% by a study conducted in Enterobacteriaceae from Burkinabe patients [17]. Moreover, few studies from other parts of world have shown different prevalence of bla CTX−M gene among isolates including 98.8% (China), 84.7% (Chile), 13.6% (Tanzania), 76% (Pakistan), 97.8% (Chad) and 81.6% (Egypt) [25,[42][43][44][45][46]. Indeed, CTX-M b-lactamases are recognized as the most widespread extendedspectrum b-lactamases (ESBLs) among clinical isolates of Enterobacteriaceae [47]. Besides, an earlier report from Nigeria has shown the predominance of CTX-M15 in wild birds and cattle in Nigeria [48] suggesting that this gene could be transferred to humans by animals. Finally, our study revealed 6.5% of TEMtype ESBL producing E. coli while no bla TEM gene has been detected in our previous study [9]. However, this value is lower than 26.2% and 28% reported in Burkina Faso and Pakistan, respectively [17,42]. The resistance to amoxicillin/amoxicillin clavulanic acid observed in the two E. coli strains (6.5%) may be mainly mediated by the production of these plasmid-encoded TEM enzymes.
Among the three class of integron, class 1 integron (58.1%) was majority characterized from the resistant strains in accordance with 56% reported in Bangladesh [41]. This result con rms those of previous studies which showed that class 1 integrons was predominantly represented in Enterobacteriaceae [49,50]. However, a previous report in Burkina Faso has shown a lower prevalence (44.4%) of Int1 [51]. On the other hand, studies reported a high prevalence of Int1 (80%) in E. coli isolated from dairy products consumed in Burkina Faso [52] and in human, animal, and food in Spain [53]. This could increase the risk of emergence and spread of MDR E. coli, since humans are always in contact with these different ecosystems, especially when there is a lack of food hygiene and sanitation. Moreover, class 1 integrons can facilitate the spread of antibiotic-resistance genes meaning that it could have public health consequences [54].
The class 3 integrons was detected in only EIEC. No class 2 integrons (Int2) was characterized from the resistant strains. By cons, 22.2% of Int2 was detected in our previous study [51]. Moreover, a study also found the presence of Int2 gene in Senegalese Shigella spp isolates [49].
Two strains of EIEC harbored both class 1 and 3 integrons. However, a previous study showed that E. coli harbored class 1 and 2 integrons simultaneously [50]. Otherwise, in the present study, one EIEC strain was resistant to aztreonam, imipenem and possess ESBL-carbapenemase phenotype. This strain was resistant to all sub-families (penicillins, cephalosporins, monobactam and carbapenems) of β-lactams antibiotic tested and also showed simultaneous presence of bla SHV , bla OXA, bla CTX−M and Int1. Indeed, strains that had this aztreonam-resistant phenotype possessed both the resistance gene [27]. Resistance to this antibiotic could be explained by genetic mutations [43]. It has been described that the coexistence of these two classes of integrons [42] and/or several genes suggests that they have integrated the same gene and give these strains a high level of resistance. However, bla TEM , bla SHV , bla OXA , bla CTX−M as well as integrons (Int1, Int2 and Int3) are involved in the antibiotic resistance of DEC, but the presence of resistant strains producing ESBL and lacking ESBL gene (bla TEM , bla SHV , bla OXA and bla CTX−M) and integron suggests that there are other mechanisms for the dissemination of antibiotic resistance in DEC strains.

Conclusions
This study highlights the important involvement of genes and integrons into multidrug resistance strains of E. coli in two main hospitals of Ouagadougou. The most important nding was the detection of four E. coli multiresistant strains producing ESBL that were resistant to imipenem, aztreonam and harbored class 1 integrons. Another important observation was the detection of two E. coli multiresistant strains producing ESBL but lacking a resistance gene and/or integrons. Our results have demonstrated the emergence and dissemination of multidrug resistant E. coli strains hosting several genes responsible for the production of ESBL in clinical isolates. Ultimately, to ght effectively against the emergence of antimicrobial resistance, an integrated surveillance network should be set up, which would be of great bene t to national antimicrobial resistance control programs.

Declarations
Ethics approval and consent to participate Permission to conduct this study was obtained from the hospital authorities of Burkina Faso and informed verbal consent was obtained from the parents of every child before samples collection. The study protocol was approved by the Ethical Committee of Burkina Faso (N° 2009-39).

Consent for publication
Not applicable.
Availability of data and materials Not applicable.