Emergence of New Delhi Metallo-β-Lactamase (NDM) Genes Detected from Clinical Strains of Escherichia coli Isolated in Ouagadougou, Burkina Faso

The emergence and spread of carbapenem resistance in Gram-negative bacilli such as Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa through the production of carbapenemases is a global phenomenon. It threatens patient care and leads to therapeutic impasses. This study aims to genotypically determine the prevalence of the most frequent carbapenemase genes among multidrug-resistant E. coli strains isolated from patients at a biomedical analysis laboratory. A total of fifty-three unduplicated E. coli strains isolated from patient samples with a multidrug-resistant (MDR) profile were subjected to polymerase chain reaction (PCR) testing for carbapenem resistance genes. This study allowed us to identify fifteen strains carrying resistance genes among the fifty-three E. coli strains. All fifteen strains produced the metallo-β-lactamase enzymes; this represents a rate of 28.30% of study strains. Among these strains, ten carried the NDM resistance gene, NDM and VIM genes were detected in three strains and VIM was identified in two strains of E. coli. However, carbapenemases A (KPC and IMI), D (OXA-48), and IMP were not detected in the strains studied. Thus, NDM and VIM are the main carbapenemases detected in the strains in our study.


Introduction
Escherichia coli (E. coli) is a versatile microorganism; it is a well-known commensal of the normal gut microbiome that can sometimes also be a very virulent and often deadly pathogen [1]. E. coli is the most frequent cause of urinary tract infections (UTI) in Burkina and is also involved in other infections such as bloodstream infections and infections of surgical wounds [2][3][4]. Te uncontrolled use of antibiotics in recent years to fght human bacterial infection has led to selection pressure in pathogen and commensal bacteria [5]. Tus, in E. coli,multidrug-resistant (MDR) strains have emerged. Indeed, many antibiotic resistance genes have been described in E. coli such as beta-lactam resistance genes TEM, SHV, CTX-M, and quinolone resistance genes (qnr) [6][7][8]. Te most clinically important resistance is that linked to carbapenems, which are betalactams of last resort used for the treatment of serious bacterial infections [9]. Several carbapenem resistance genes have been described in E. coli [10]; NDM-1 (New Delhi metallo-beta-lactamase-1) is the most recently discovered transferable molecular class B. It was frst described in Klebsiella pneumoniae and E. coli isolated in Sweden in 2008 from a patient transferred from a New Delhi hospital [11].
Enterobacteriaceae are responsible for a large proportion of nosocomial infections and are associated with signifcant morbidity and mortality; the emergence and dissemination of resistance to carbapenems pose a major public health problem. So, information of carbapenemase-producing bacteria study is limited in Burkina Faso. Knowledge of the types of resistance genes will guide the choice of appropriate antibiotic therapy. It will make it possible to formulate recommendations on the use of antibiotics as well as the implementation of infection control strategies. Tis study aimed to determine genotypically the prevalence of genes encoding resistance to carbapenems in E. coli species. Tis involved looking for carbapenemases: Klebsiella pneumoniae carbapenemase (KPC), Imipenem-hydrolyzing-β-lactamase (IMI), active on imipenem (IMP), New Delhi metallo-β-lactamase (NDM), Verona integron-encoded for metallo-β-lactamase (VIM), and oxacillinase (OXA-48) in E. coli multidrug-resistant isolated in Ouagadougou, Burkina Faso.
To carry out the study, strains presenting a multidrugresistance profle were collected and carbapenem sensitivity test was conducted on Mueller-Hinton agar (MHA) using disc method. Ten, bacterial DNA extracts were made for the PCR reaction on strains showing resistance or reduced sensitivity to carbapenems.

Bacterial Isolates.
Tis was a retrospective study of clinical strains collected after routine analysis. A total of 53 unduplicated strains of E. coli multidrug-resistant (MDR) were collected from cultured urine and pus specimens at Schiphra Hospital Medical Analysis Laboratory, from inpatient and outpatient specimens (Figure 1), from February to September 2020. Most of the strains collected came from urine culture and only four strains were isolated from pus (E. coli P80, E. coli P66, E. coli P46, E. coli P49) culture. Clinical strains were identifed by standard bacteriological methods using Gram stain and biochemical method using API 20E kits (bioMérieux, Marcy-l'Étoile, France) gallery. Pure strains were inoculated into Luria Bertani broth supplemented with 20% glycerol and stored at −20°C for subsequent tests and analyses.

Antimicrobial Susceptibility
Testing. Antibiotic sensitivity was performed on Mueller-Hinton agar medium (MH) according to the CA-SFM/EUCAST guidelines [12]. A bacterial suspension corresponding to 0.5 McFarland was inoculated by swab on MH medium, then the antibiotic discs were deposited. After 18-24 hours of incubation at 37°C, the reading was carried out and the strains were classifed susceptible, intermediate, or resistant with respect to the critical diameter according to CA-SFM/EUCAST guidelines.
Following antibiotics were tested on the strains in routine examination: Fosfomycin, Norfoxacin, Gentamicin, Cotrimoxazole, Ciprofoxacin, Levofoxacin, Ceftriaxone, Cefxime, Chloramphenicol, Cefuroxime, Imipenem, Amikacin, Ticarcillin-Clavulanate, Ceftazidime, Meropenem, Cefepime, Ticarcillin, Aztreonam, Netilmicin, Nitrofuran, and Amoxicillin-Clavulanate. Te susceptibility profle to the diferent antibiotics of all the strains is presented by Kaboré et al. [4]. Tese antibiotics were used in our study because they are the frst-line antibiotics used for bacterial infections. However, carbapenems were used in the study to determine resistance to these so-called last-resort antibiotics for Gramnegative infections. After the collection of the bacterial strains, the antibiogram was performed on the strains with carbapenems, imipenem, meropenem, and ertapenem (company Lioflchem, Italy). When resistance to carbapenems has been found, the Imipenem-EDTA and Imipenemboric acid test were performed for observation of the inhibition diameter, this for phenotypic detection of metallobeta-lactamase and carbapenemase class A [13].

Molecular Characterization for Detection of
Carbapenemase-Encoding Genes

DNA Extraction.
A total of 2-3 identical colonies of E. coli were resuspended in 250 μL of distilled sterile water in 1.5 mL Eppendorf tube. Tis was boiled at 100°C for 10 min and immediately frozen at −20°C for 10 min, centrifuged at 13000 rpm for 5 min, and the supernatant containing DNA was stored at −20°C for further using [14][15][16][17]. Te presence of DNA was assessed by qualitative analysis in agarose gel electrophoresis.

Molecular Identifcation of Carbapenemase Genes.
All strains of E. coli were tested for PCR using oligonucleotide primers specifc for the carbapenem resistance genes listed in Table 1. Genes were amplifed by polymerase chain reaction (PCR) on a Mastercycler nexus gradient (Eppendorf, fexlid). Reaction mixture consisted of a total volume of 25 μL; composed of 4 μL of Master Mix (Inqaba Biotec), 2 μL of primers (Forward and Reverse), 17 μL of nuclease-free water, and 2 μL of DNA extract. PCR reagents were provided by Inqaba Biotec West Africa, Nigeria. Amplifcation reactions were performed in a thermal cycler (Mastercycler nexus gradient) according to the following program: initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 45 seconds, annealing at specifc primer temperature (Table 1), extension at 72°C for 1 minute, and fnal extension at 72°C for 10 minutes for 35 cycles. Pseudomonas aeruginosa ATCC 27853 was used in the PCR cycle and used as a negative control for the carbapenemase genes sought.
After PCR amplifcation, 4 μL of each reaction were separated by 1.2% agarose gel electrophoresis for 120 min at 80 V in TAE 1X bufer in the migration tank (ENDURO ™ GEL XL). DNA was stained with ethidium bromide (1 μg/ mL) and amplifed DNA bands were visualized using a UV transilluminator (UVP PhotoDoc-lt Imaging System).

Results and Discussion
3.1. Antibiotic Testing. All the strains were tested on several classes of antibiotics including beta-lactams and proved to be multiresistant (MDR) [4]. Te phenotypic profle of carbapenemase-producing strains detected by polymerase chain reaction is shown in Table 2. Figure 2 shows the phenotypic profle of metallo-betalactamase production observed when these strains are carbapenemases-producing type MBL.
Inhibition zone ≥7 mm with the Imipenem-EDTA disk was compared to the Imipenem disk alone and was considered as MBL positive [24,25].
It is difcult to demonstrate resistance to carbapenems by the disc difusion method. In our study, only two (E. coli 1318 and E. coli 919) strains showed resistance by disc difusion method, while the PCR technique revealed 15 strains showing the presence of carbapenemase genes. In fact, according to the CA-SFM [12], any strain with reduced sensitivity to at least one of the carbapenems must therefore   International Journal of Microbiology be considered suspicious of carbapenem production. Furthermore, the CA-SFM recalls that Ertapenem is the carbapenem with the best sensitivity for the detection of carbapenem-producing strains. Tus, any strain showing reduced sensitivity to Ertapenem (MIC >0.5 mg/L or an inhibition diameter (10 μg/ml disk) <25 mm) by the agar difusion test can be subjected to the screening algorithm for carbapenemase-producing strains. Given the results of sensitivity to carbapenems, it should not be assumed that the strains (E. coli 1473, E. coli 1476, and E. coli 1321) can be detected as carbapenemase producers because these strains present a phenotypic sensitivity to carbapenems and to Ertapenem considered carbapenem of choice for the detection of carbapenemase-producing strains. Te average age of the patients is 60.21 years with extremes of 11 years and 85 years. Male sex is predominant at 64.28% (n = 9) with extreme ages 60 years and over except one male patient who is 42 years old. At these ages, infections are more frequent with recurrent antibiotic treatments resulting in selection pressure for resistant strains.

Carbapenemase Genes.
Among the strains of E. coli, including 49 from urinary infections and 4 from pus, we found ten strains whose PCR reaction products showed bands corresponding to the bands of the NDM genes at 488 bp, which represents a rate of 18.87% and PCR products from two strains showed bands at 212 bp corresponding to VIM. However, we found that the PCR products of three strains showed two bands at both 488 bp and 212 bp (NDM and VIM); these are E. coli 1318, E. coli 514, and E. coli 583 strains (Table 3). Indeed, the PCR did not reveal the presence of any bands for the genes corresponding to base pairs 481 (IMI), 796 (KPC), 744 (OXA-48 likes), and 432 (IMP). No bands corresponding to a carbapenemase gene were observed with the PCR products of the DNA extracts of the  Figure 3 shows the band profle of the NDM resistance genes visualized with the UVP PhotoDoc-lt imaging system after migration, and Figure 4 shows the band profle of the VIM resistance genes. Table 3 shows the result of the genotypic investigation of the resistance genes sought, with (−) indicating that the gene sought was not detected and (+) indicating that the gene sought was detected.
Te presence of carbapenemase-producing bacteria in hospitals poses a serious challenge. Te problem with carbapenemases is that most of the resistance genes are located on mobile elements and are therefore easily transferred from one bacterial species to another [26][27][28]. Indeed, out of 53 multidrug-resistant E. coli strains, we identifed 15 strains producing metallo-beta-lactamase genes, of which ten harboured the NDM gene, two strains harboured the VIM gene, and three harboured both genes at the same time (NDM and VIM). All these strains came mainly from urinary infections (49 strains were of urinary origin and 4 came from pus). Te rate of carbapenemase-producing E. coli among the multidrug-resistant strains was 28.30%, which confrms the multidrug-resistant nature of these strains. In our study, only carbapenemase B (NDM and VIM) were detected in E. coli strains, which could explain their rapid difusion within the bacterial strains. Other resistance genes were not researched and the strains could harbour these genes, hence their multidrug-resistance; since these genes have already been described in E. coli in Burkina Faso (TEM, SHV, CTX-M,. . .), so genes for resistance to other families of antibiotics (quinolones, aminoglycosides, etc.) were not sought. However, previous studies had revealed a high rate of extended-spectrum beta-lactamases among clinical strains in Burkina Faso [6,7,29]. In West Africa, the prevalence of ESBL varies from one country to another; thus, rates of 49.4% and 63.4% to 96% have been reported respectively in Ghana and Mali in hospitals and in the community [30]. Our study revealed the presence of DNA bands at 488 bp for NDM and 212 bp for VIM of the PCR reaction products of E. coli strains. Indeed, the presence of carbapenemase genes of the NDM, VIM, and OXA type had been detected in strains of E. coli and other Gram-negative bacilli [29]. An OXA-181-like carbapenemase was detected in four species of E. coli for the frst time in Burkina Faso [31]. Tese authors have been able to demonstrate the plasmids on which these genes are located, namely IncX3 for the OXA-181 type genes and IncF for the NDM-1 genes. In West Africa, few studies have been reported on the resistance of carbapenemase-producing Gram-negative bacilli. However, some case studies in Togo, Mali, and Nigeria have    International Journal of Microbiology reported the presence of OXA and NDM genes [32][33][34]. NDM, OXA, and VIM genes have been reported in most African countries [35]. Te movement of populations for economic and health reasons could explain the rapid spread of the NDM gene around the world; indeed, travelers to endemic areas are at risk of exposure to resistant pathogens and are likely to return colonized [36,37]. Described in Klebsiella pneumoniae isolated from a Swedish patient of Indian origin with a urinary tract infection in New Delhi, India, and designated NDM-1 [12], more than twenty NDM variants have been described to date [38]. Te genetic variability of the NDM gene could explain its worldwide distribution. Asian continent serves as the major reservoir of NDM producers, with around 58.15% abundance of NDM-1 variant distributed mostly in China and India [39].

Limit of the Study.
We did not have the opportunity to confrm the expression of carbapenemase genes by DNA sequencing technique.

Conclusion
Our study highlighted the presence of resistance genes NDM and VIM among the clinical strains of E. coli in Burkina. NDM and VIM enzymes hinder the action of beta-lactams and other antibiotics, resulting in therapeutic impasses. Terefore, increased surveillance should prevent the spread of these resistance genes, which are a major concern worldwide. Finally, a structural study of these enzymes could make it possible to develop new molecules in association with beta-lactams to combat these resistances.

Data Availability
Te data used to support the fndings of this study are available from the corresponding authors upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Investigation, analysis, and original draft preparation are realized by KB, HSO, AGO, and SB. Paper is revised by OZ, FT, KJZ, and BZ. Te validation of results from investigation and review of paper are supervised by YT, IS, and AS. All authors have read and approved the fnal manuscript.