Multi-resistance to carbapenems by the production of Imipenemase (IMP)-types carbapenemases in Gram-negative bacilli in Burkina Faso

The growing prevalence of Imipenemase (IMP) metallo-beta-lactamases (MBL) producing strains of Gram-negative bacilli (GNB) pathogens is a real concern for clinicians in the light of therapeutic impasses driven. However, resistance genes encoding these enzymes are hardly documented in Burkina Faso. This study aims to show carbapenem-resistance mediated by the production of IMP-type carbapenemase in GNB clinical strains collected in Ouagadougou, Burkina Faso. Strains resistance profile to imipenem, meropenem, ertapenem, doripenem and aztreonam was determined by the disk diffusion method. Classical polymerase chain reaction (PCR) was carried out to detect β-lactamase ( bla ) gene of IMP in resistant strains. Out of 158 GNB collected, 91 (57.6%) were resistant to at least one of the carbapenems and/or to aztreonam. The highest prevalence of resistant strains was observed in Escherichia coli (45.1%; n=41) and Klebsiella pneumoniae (26.5% n=24). Among 32 resistant strains bla IMP gene positive (35.2%), Escherichia coli was the predominant species carrying resistance gene (18.7%, n=17/91). The findings strengthen the scarce existing scientific data on antimicrobial resistance mediated by metallo-beta-lactamases (MBL) in Burkina Faso.


INTRODUCTION
Antibiotic therapy aims to kill pathogen bacteria.Unfortunately, these bacteria to protect themselves develop resistance to antibiotics used to treat them.Bacteria resistant to several types of antibiotics are sometime referred to as multi-resistant.Increased and inappropriate use of antimicrobial drugs promotes the emergence of antimicrobial resistant bacterial strains and related infections (Masoud et al., 2021;Kim et al., 2023).Some of those bacteria have become resistant to several antibiotics, including carbapenems and third-generation cephalosporins (Palacios-Baena et al., 2021;Arumugham et al., 2022).The emergence and spread of multidrugresistant Gram-negative bacilli (MDR) has become a major public health concern worldwide (Dembele et al., 2020).The spread of MDR Gram-negative bacilli is increasingly reported in both hospital and community settings worldwide (Manenzhe et al., 2015).In the last decade, an alarming increase in the prevalence of carbapenemase-producing Gram-negative bacilli of serious nosocomial infections has been shown worldwide (Bourafa et al., 2018;Haji et al., 2021).Indeed, carbapenems were considered as last line drugs for controlling multidrug-resistant Gram-negative pathogens including extended-spectrum beta lactamases (ESBL)producing Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumanii (Haji et al., 2021;Ibrahim et al., 2021;Dikoumba et al., 2023).However, overprescribing of carbapenems has led to emergence and global dissemination of carbapenem-aresistant organisms, including carbapenem-resistant Enterobacteriaceae, carbapenem-resistant P. aeruginosa and carbapenem-resistant A. baumanii (Aruhomukama et al., 2019;Haji et al., 2021) which are on World Health Organization (WHO) list of critical priority 1 pathogens of urgent need for new antimicrobial development (Tacconelli et al., 2018).Carbapenemase enzymes are the most common mechanism by which resistance to carbapenems occurs in these GNB (Shaker et al., 2018;Aruhomukama et al., 2019;Dziri et al., 2020;Haji et al., 2021).Several types of carbapenemases have been described.

Sample and bacterial isolates identification
This study was conducted in Ouagadougou, Burkina Faso.This was a cross-sectional study with retrospective data collection of 158 bacterial strains which were collected from September 2018 to October 2018 and from September 2022 to August 2022 in Bacteriology Department of the Laboratories of University Hospital Center of Tengandogo (CHU-T) and of Saint Camille Hospital of Ouagadougou (HOSCO) in Burkina Faso.These bacterial samples were GNB strains resistant to at least one third generation cephalosporin and/or aztreonam, including 130 isolates from HOSCO and 28 others from CHU-T.They were isolated in the following specimens: Urine, stool, pus, blood cultures, vaginal and vulvar swabs and peritoneal fluids collected from outpatients and hospitalized patients.API 20E gallery tests (BioMerieux S.A., Marcy Etoile, French) were used for bacterial species identification.

Molecular identification of gene encoding IMP carbapenemase
All isolates resistant to at least one carbapenem and/or aztreonam were screened by conventional PCR (Polymerase Chain Reaction), using specific oligonucleotide primers (Table 1) to gene encoding beta-lactamase of bla IMP family.Reaction mixture of 20 μL was composed of 4 µL Firepol® Master Mix 5X, 0.5 µL of each primer (Forward and Reverse), 14 µL of PCR water and 1 µL of DNA extract.Amplification reactions were performed using GeneAmp System PCR 9700 Thermal Cycler (Applied Biosystems, California, USA) according to PCR program shown in Table 2.After amplification, 6 to 8 μL of each PCR product were separated by 1.5% agarose gel electrophoresis at 100 volts for 35 min in TAE 1X buffer mixed to ethidium bromide (1 μg/mL) in migration tank.A molecular weight marker, DNA ladder (Solis Biodyne, Estonia) 100 pb was used as a reference.Amplified DNA bands were visualized using a UV transilluminator (E-Box Vilber).
The distribution of bacterial species according to antibiotic resistance patterns are shown in Table 4.The resistance rates observed were: 94.5% (n=86) for aztreonam, 44.0% (n=40) for ertapenem and 22.0% (n=20) for each imipenem, meropenem and doripenem.For ertapenem, the carbapenem with the highest resistance, E. coli strains recorded the highest level of resistance (20.9%, n=19/91) followed by K. pneumoniae (11%, n=10/91) and P. aeruginosa (6.6%, n=6/91).On the other hand, the susceptibility of the isolates to antibiotics revealed that almost all strains were highly resistant to aztreonam.

Molecular detection of gene encoding IMP carbapenemase
Molecular characterization of gene encoding IMP-type carbapenemase performed by conventional PCR, using specific primers (Table 1) revealed that, out of 91 resistant isolates, 32 strains (35.2%) harbored blaIMP gene as showing DNA bands appeared approximatively at 488 pb (Figure 2).

DISCUSSION
Carbapenems over-prescribing has led to emergence and global increased dissemination of carbapenemresistant bacteria which reduced the effectiveness of these antibiotics in the treatment of related infections    100) n = number of resistant strains of each bacterial species; % = proportion of resistant strains of each bacterial species, HOSCO = Saint Camille Hospital Center in Ouagadougou; CHU-T = Tengandogo University Hospital Center.(Aruhomukama et al., 2019;Haji et al., 2021;Das, 2023).An alarming increase in the prevalence of carbapenemase-producing Gram-negative bacilli of serious nosocomial infections has been reported worldwide (Bourafa et al., 2018;Haji et al., 2021).This current study was undertaken to show   carbapenem resistance in GNB by production of IMPtype carbapenemase from urban hospitals centers in Ouagadougou, Burkina Faso.Overall 158 GNB strains collected in HOSCO and in CHU-T, 57.6% (n=91) of them were carbapenem-resistant including E. coli (45.1%, n=41) and K. pneumoniae (26.5%, n=24) which were the predominant resistant species followed by P. aeruginosa (9.9%, n=9).In Ghana, Owusu et al. ( 2023) reported these similar findings, where E. coli and K. pneumoniae were the predominant GNB species at 46% (n=83) and 17% (n=30), respectively.Furthermore, studies conducted in other countries have reported both same species with the most carbapenem-resistant strains (Haji et al., 2021;Armin et al., 2023).The results are in agreement with the study in Burkina Faso by Kabore et al. (2022) who reported that GNB is mainly more resistant to antibiotics including carbapenems were E. coli and K. pneumoniae, but at higher rate (82.69%) and lower rate (9.62%) respectively.In contrast, Balkhair et al. (2023) found a lowest resistance rate to carbapenems for E. coli (2.9%) and a higher carbapenem level resistance at 46.4 and 29.9% respectively for K. pneumoniae and P. aeruginosa.
One the other hand, this study carbapenem-resistant strains prevalence (57.6%, n=91/158) was higher than those (30.9,37 and 27.7%) observed respectively by Haji et al. (2021), Armin et al. (2023) and Balkhair et al. (2023).Indeed, any strain exhibiting reduced sensitivity to at least one of carbapenems and/or aztreonam was categorized resistant and therefore was suspicious of carbapenemase production.This fact and sample size would explain the prevalence of carbapenem-resistant strains.The majority of the E. coli, K. pneumoniae and P. aeruginosa isolates were originated from urine (50.5%) collected mainly from female patients (51.6%).Also, Haji et al. (2021) observed urine samples were the dominant type (62%) from which these species were isolated.Regarding antibiotic resistance profile, the strains showed slightly high resistance level to carbapenems (Table 4).Thus, imipenem with 22.0% as resistance rate, is currently one the most effective carbapenem as reported by Masoud et al. (2021).However, Haji et al. (2021) have recorded slightly higher resistance rates for imipenem (41%) and meropenem (40%) and a slightly low resistance level for ertapenem (36%).In this study, carbapenem resistance profile was carried out by an automated method (Hu et al., 2019) which improve resistance detection.Whereas, in this study phenotypic detection of carbapenem-resistant strains has been performed using manual disk diffusion method (EUCAST/CASFM, 2022), by which it is difficult to demonstrate carbapenem resistance (Kabore et al., 2023).All bacterial isolates resistant to carbapenem and/or to aztreonam therefore suspicious of carbapenemase production were screening by molecular approaches to detect IMP gene.In this current study, blaIMP gene was found in 35.2% (n=32/91) of carbapenem-resistant strains, while in 64.8% (n=59/91) other strains none resistance IMP gene was found (Table 5).A previous of Dembele et al. (2021) in Burkina Faso has demonstrated the presence of blaIMP-2 gene in Imipenemresistant E. coli strains isolated from children with diarrhea in rural settings.Whereas, in this study, carbapenemase-IMP encoding gene was identified not only in E. coli, but also in other enterobacteria (Table 5) and then in P. aeruginosa recovered from children or adult patients in urban health centers in Ouagadougou, Burkina Faso.
IMP gene was recorded at a slightly high prevalence (35.2%, n=32/91) and more than half of gene positive strains (18.7%, n=17) were Escherichia coli.These findings are comparable to those reported in Iraq, where detection rate of blaIMP gene was 43% (n=23/53) in clinical GNB strains, and this gene most prevalent in Escherichia coli (50%) (Haji et al., 2021).Our carbapenemase-IMP gene frequency (35.2%, n=32/91) was highest than those recorded in previous studies in Sudan (26.4%) (Adam et Elhag, 2018), in Egypt (11.8%) (Abbas et al., 2019) and in Iran (13%) (Armin et al., 2023).This increasing detection rate of blaIMP comparing to frequencies of these previous studies would suggested a currently worrying spread of carbapenem-resistant GNB producing IMP-type carbapenemase (Li et al., 2023).In addition, to the best of our knowledge, our study is one of the first in our country to record detection of blaIMP gene in Pseudomonas aeruginosa carbapenem resistant clinical strains with a prevalence of 3.3% (Table 5) in health centers of Ouagadougou.Until then, Kabore et al., in their study, had been limited to the phenotypic detection of MBL in Imipenem-resistant Pseudomonas aeruginosa clinical strains .This detection rate of blaIMP gene in Pseudomonas aeruginosa is certainly underestimated given the relatively small sample size of our study.Thus, our study firstly reporting the circulation of Pseudomonas aeruginosa producing IMP-type carbapenemases in health centers of Ouagadougou, Burkina Faso.
In contrast, out of 64.8% (n=59/91) resistant strains, none strain has harbored blaIMP gene encoding IMP-type carbapenemase although some of them (12.1%, n=11/91) were resistant to all carbapenems and aztreonam tested.This fact could be explained by the existence of other resistance mechanisms in our isolates, such as the production of ESBLs or other carbapenemases and defects in cell wall permeability.Indeed, in a previous study conducted in 2022 by Tiemtore et al. (2022) in Burkina Faso, blaGES gene encoding Guiana Extended-Spectrum (GES) carbapenemase has been detected in Imipenem-resistant enterobacteriaceae strains included in this study.Thus, eventual presence of GES-2 variant, exhibiting an increased hydrolysis of carbapenems (Poirel et al., 2001), in our bacterial strains, might justified carbapenem resistance of IMP gene-negative isolates.Interestingly, the encoding-carbapenemase genes, such as blaNDM, blaVIM and bla KPC were found in GNB clinical isolates as previously reported in other studies in this country (Dembele et al., 2021;Kabore et al., 2023;Ouattara et al., 2023;Bambara et al., 2023).These carbapenem resistance genes not researched in this study could also explained the finding.Further, non-enzymatic resistance mechanisms to carbapenems such as defecting in wall cell permeability through modification or loss of porins (Zango et al., 2019;Eichenberger and Thaden, 2019;Çekin et al., 2021;Onishi et al., 2023) and overexpression of efflux pumps (Lee et al., 2021;Onishi et al., 2023) would explained the result.
Finally, IMP gene was detected in 9.9% (n=9/91) strains which were susceptible to all carbapenems except aztreonam.This presence of MBL gene encoding IMP enzyme in GNB isolates sensitive to carbapenems finding in this study was reported in previous studies in other countries with various frequencies including NDM and VIM (Anoar et al., 2014;Adam and Elhag, 2018).These results corroborate hypothesis that, ordinarily, MBL genes presence among carbapenems sensitive strains indicate that there might be hidden MBL genes not detected by phenotypic tests, leading to the silent spread of these genes in the hospitals and the community (Adam and Elhag, 2018).

Conclusion
Carbapenem-resistant Gram-negative bacilli are a growing threat to public health worldwide.The study aimed to determine carbapenem multi-resistance mediated by IMP carbapenemase production in GNB at Saint Camille Hospital of Ouagadougou (HOSCO) and University Hospital Center of Tengandogo (CHU-T) in Ouagadougou, Burkina Faso.The GNB strains exhibited a high resistance level to antibiotics tested with a prevalence of 57.6%.This study revealed a slightly high prevalence of blaIMP, gene encoding IMP-type metallobeta-lactamase in carbapenem-resistant strains.These results confirm the presence of GNB resistant to antibiotics producing IMP-type carbapenemases at HOSCO and CHU-T in Ouagadougou, Burkina Faso.The findings of this study strengthen the scarce existing scientific data on antimicrobial resistance mediated by metallo-beta-lactamases in Burkina Faso.The study recommended an urgent implementation of an antibiotic resistance surveillance system combining clinical aspects of infections related to bacteria producing MBL and other carbapenemases, in order to prevent, monitor and control the spread of antimicrobial resistance gene in our country.Further studies on sequence analysis of IMP gene amplicons are also to know different IMP variant profile in carbapenem-resistant GNB in Burkina Faso.

Figure 1 .
Figure 1.Petri dishes showing resistance of strain to antibiotics tested.
; n= strain number of each bacterial species; n' = number of resistant strains of each bacterial species to antibiotic overall of the total of strains; %= percent correlated to the total number of resistant strains bacterial; IPM= Imipenem, MRP= Meropenem, ERT= Ertapenem, DOR = Doripenem, ATM = Aztreonam.

Table 1 .
Primers used for bla IMP gene detection.

Molecular detection of gene encoding IMP-type carbapenemase Extraction of bacterial DNA Bacterial
(Dashti et al., 2009)m isolated colonies obtained from previously strains stored in Luria Bertani storage medium, which were awakened by culturing on MH agar for 18 to 24 h at 37°C.Bacterial DNA extraction was done by the boiling method(Dashti et al., 2009)with few modifications.A total of 2 to 3 identical colonies were suspended in 200 μL of distilled sterile water in 1.5 mL labeled Eppendorf tube.The resulting suspension was boiled at 100°C for 15min in water bath (MEMMERT, Rost fret) to release bacterial genetic material and then centrifuged (NF 048 centrifuge) at 12000 rpm for 10 min to remove genetic material from others debris.The supernatant containing DNA was transferred to a new Eppendorf tube.DNA extract was assayed for quantity and purity, using NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, United States) and then stored at -20°C for further usage.

Table 2 .
PCR program for bla IMP gene detection.

Table 3 .
Frequencies of bacterial resistant strains according to area sampling.

Table 4 .
Distribution of bacterial species according to antibiotic resistance patterns.

Table 5 .
Distribution of IMP gene according to resistant bacterial species.= number of resistant strains of each bacterial species harboring or not resistance gene; '+'indicating gene detected; '-'= indicating gene not detected; %= percent correlated to the total number resistant strains bacterial. n