Objective: Class B carbapenemases are bacterial enzymes that catalyze the hydrolysis of
β-lactam core antibiotics, except for monobactams. The objective of this study was to identify the carbapenemase gene
blaIMP in the genus
Klebsiella at the Charles De Gaulle Pediatric University Hospital (CHUP-CDG) of Ouagadougou, Burkina Faso.
Methods: The study involved 17 bacterial strains responsible for human infection and isolated from various biological samples during the period from 2009 to 2013. The strains were tested for antimicrobial susceptibility to cefotaxime, ceftazidime and imipenem using the Mueller-Hinton agar diffusion method. The carbapenemases resistance genes were detected by conventional PCR using specific primers at the molecular biology laboratory of CERBA/LABIOGENE, Ouagadougou, Burkina Faso.
Results: The antibiotic susceptibility test showed high resistance of the 17
Klebsiella isolates tested to cephalosporins. A high cefotaxime-resistance rate (82.35%) and ceftazidime-resistance rate (88.23%) was found among the strains tested against 11.76% resistance rate for imipenem. Analysis of PCR products by gel electrophoresis revealed 4 strains (23.53%) with
blaIMP-type gene.
Conclusion:
Klebsiella is a well-known bacterium in clinical practice. The present study demonstrated the
blaIMP-type gene in cephalosporin-resistant strains of
Klebsiella at CHUP-CDG. More effective monitoring and treatment solutions are needed to prevent the spread of these resistant strains.
Klebsiella is a genus of flowering plants in the family Enterobacteriacea. It includes several species which are: K. pneumoniae, K. oxytoca, K. ornithinolytica, K. planticola, K. terrigena, K. granulomatis, K. variicola, K. singaporensis [1] . The species K. pneumoniae is often implicated in human infections. It is an opportunistic agent of community and nosocomial infections such as urinary tract infections, respiratory tract infections and wound infections. K. pneumoniae is a commensal bacterium of the gastrointestinal and respiratory tracts. It is also present in the environment [2] . Klebsiella oxytoca is an opportunistic pathogen in humans and is increasingly associated to nosocomial infections, particularly in immunocompromised individuals [3] . Klebsiella species are naturally resistant to penicillin such as ampicillin, ticarcillin and piperacillin. Wild strains are susceptible to antibiotics active on gram-negative bacteria such as aminoglycosides, fluoroquinolones, sulfonamides, fosfomycin, colistin, furans, cephalosporins and carbapenems [4] . However, the emergence of β-lactamases, enzymes capable of hydrolyzing β-lactams, is increasingly described in clinical isolates from Klebsiella which is a public health concern. [3] According to Ambler’s classification, β-lactamases are subdivided into four groups (A, B, C and D) based on their amino acid sequences [5] .
KPC Class A carbapenemases and zinc-dependent Class B metallo-β-lactamases (MBL), primarily VIM, IMP and NDM metallo-β-lactamases, are the most clinically important carbapenemases in Enterobacteriacea [5] [6] . These β-lactamases hydrolyze penicillins, cephalosporins and carbapenems, although significant variations in hydrolytic efficiency exist even between enzymes of the same type [7] . The emergence of carbapenemases-producing Enterobacteriaceae, particularly Klebsiella, which produces KPC, VIM, IMP and NDM carbapenemases, is a public health concern [7] . Carbapenemase genes have been described in several epidemiological studies in Europe, North America and Asia [8] [9] . In Africa, there is little data available on carbapenemase genes. A previous study reported the presence of carbapenemase-producing bacteria at rates of 74% in North Africa; 12% in Southern Africa; 90% in South Africa; 8% in West Africa (7/83) and 6% in East Africa [10] . In Burkina Faso, a study on germ resistance revealed the presence of Verona integrin-encoded metallo-β-lactamase (VIM) and imipenemase (IMP-2) reaching a rate of 40% each [11] . However, no information is available on the different types of carbapenemase genes harbored by Klebsiella species.
The main objective of this study was to characterize the IMP-type carbapenemase gene carried by Klebsiella species, isolated between 2009 and 2013 at the Centre Hospitalier Universitaire Pédiatrique Charles de Gaulle, Ouagadougou.
2. Material and Methods2.1. Bacterial Strains
Bacterial isolates were collected consecutively between 2009 and 2013 from children at the CHUP-CDG of Ouagadougou, Burkina Faso [12] . They were isolated from various clinical specimens such as urine, pus, and CSF from diverse units of the hospital and conserved in the biobank of CERBA/LABIOGENE. Once identified, the isolates were preserved at −80˚C in Tryptic Soy Broth containing glycerol 15% (v/v). From June to September 2021, the 17 Klebsiella strains were selected from the biobank for antibiogram testing and screening for the blaIMP gene.
2.2. Antimicrobial Susceptibility Testing
The antimicrobial susceptibility test was performed by the disk diffusion method on Mueller Hinton (MH) agar using pure Klebsiella colonies according to the recommendations of the Antibiogram Committee of the French Society of Microbiology [4] . The antibiotic disks used were Cefotaxime, Ceftazidime, and Imipenem.
2.3. DNA Extraction
The boiling method was used to extract DNA [13] . The strains were reactivated by culture on the MH medium for 18 to 24 h and then in liquid LB medium. The bacterial suspensions were then immersed in a water bath at 100˚C for 15 minutes to release the bacteria’s genetic material. After that, a centrifugation of 10 min at 12,000 rpm was performed and the supernatant containing the released DNA was transferred to a new Eppendorf tube. Total DNA quantification and DNA purity based on absorbance ratios A260/A280 and A260/A230 were determined for each sample using NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
2.4. Antibiotic-Resistant Gene Amplification
The BlaIMP genes were detected by PCR with the following specific primers: IMPF; 5’CATGGTGTGGTGCTTGT3’ and IMP-R; 5’ATAATTTGGCGGACTTTGGC3’, with expected amplicon size 488 bp [14] . The PCR amplification was performed in 20 μl volumes containing 4 μL of GREEN PCR Master Mix 5X, 0.5 μL of each primer, 14 μL of sterile PCR water and 1 μL of DNA template in a Gene Amp 9700 PCR System thermocycler (Applied Biosystems, California, USA). The PCR conditions consisted of denaturation at 96˚C for 5 min, then 30 cycles of denaturation for 30 s at 95˚C, annealing for 30 s at 54˚C and extension for 30 s at 72˚C following by a final extension for 7 min at 72˚C.
2.5. Electrophoresis
PCR products were loaded on a 1.5% agarose gel with 0.1% ethidium bromide in 1X Tris-acetate EDTA (TAE) buffer, separated for 25 min at 100 millivolts, and visualized under ultraviolet light using GeneFlash (Syngene, Bio-Imaging, UK). A 100 bp DNA ladder was used to visualize fragments size. PCR mix with no DNA template was included as a negative control.
2.6. Statistical Analysis
The collected data was entered into Microsoft excel spreadsheet, cleaned, and imported into STATA software package v.14.0 (Stata Corporation, College Station, TX) for analysis. Data were described as percentage (%) and frequency of occurrence for categorical variables.
2.7. Ethical Considerations
The institutional ethic committee of CERBA/LABIOGENE reviewed and approved the study protocol.
3. Results3.1. Bacterial Isolates
From 2009 to 2013, a total of 17 Klebsiella isolates were collected from the different wards of CHUP-CDG, Ouagadougou, Burkina Faso. Urine provided the majority of the Klebsiella clinical isolates (n = 10, 58.82%), followed by pus (n = 6, 35.30%) and cerebrospinal fluid (n = 1, 5.88%).
3.2. Antimicrobial Susceptibility Testing
A total of 17 Klebsiella isolates from clinical specimens were tested for antimicrobial susceptibility to cefotaxime (CTX), ceftazidime (CAZ) and imipenem. The results revealed a high cefotaxime (14/17; 82.35%) and ceftazidime (15/17, 88.23%) resistance rate against 11.76% (4/17) resistance rate for imipenem. It is noteworthy that 8 out of 10 Klebsiella pneumoniae and all 4 K. oxytoca were resistant to CTX and CAZ (Table 1).
3.3. Molecular Characterization of bla<sub>IMP</sub> Genes
The blaIMPmetallo-β-lactamasesgenes were sought by PCR in 17 Klebsiella isolates from CHUP-CDG and 3 (4/17 or 23.53%) of them yielded the amplicon of blaIMP gene (Figure 1). Among these 4 blaIMP-carrying strains, 3 were Klebsiella oxytoca and 1 was Klebsiella pneumoniae (Table 2).
4. Discussion
Except for aztreonam, MBLs may confer resistance to carbapenems and all other β-lactam [15] . MBL types of VIM, IMP and NDM are the most common in the world [16] . These enzymes are mainly found in gram-negative bacilli. MBL IMP has been reported primarily in P. aeruginosa, Acinetobacter spp and several species of Enterobacteriaceae, including E. coli, K. pneumoniae, Klebsiella oxytoca, E. cloacae and Citrobacter spp [17] . Bacteria carrying the blaIMP gene are referred to as carbapenem-resistant Enterobacteriaceae (CRE). The present study was
focused on the identification of carbapenem-resistant Klebsiella collected from 2008 to 2013. The antimicrobial susceptibility test revealed significant resistance to third generation cephalosporins (C3G). The resistance rate was 82.35% for cefotaxime (CTX) and 88.23% for ceftazidime (CAZ). C3G resistance has been reported in Benin [18] (97.5% for CAZ and CRO), Togo [19] (97.28% for CAZ, 97.16% for CRO and 100% for CTX) and Burkina Faso [20] (76.6% for CTX, 75% for CRO, 58.3% for CAZ). Resistance to third generation cephalosporins has been reported in Klebsiella pneumoniae strains in Bangladesh [21] (ceftriaxone 72.8%, and ceftazidime 75.3%) in Iran [22] (ceftriaxone 73% and ceftazidime 72%). The high rate of resistance to third generation cephalosporins observed in this study could be due to the presence of certain resistance genes. These genes are located on the bacterial chromosome or on mobile genetic elements such as plasmids or transposons that can be transferred from one strain to another of the same species, but also between two closely related species [23] [24] .
In our study, carbapenem (35.28%) was weakly resistant, as were those in Bangladesh [21] (meropenem for 44.7%) and Iran (meropenem 43.4%). It has been very sensitive in India [25] (100% for imipenem), Ghana [26] (100% for meropenem) and Iran [27] (imipenem for 58.2%; meropenem for 64.3%; doripenem for 64.3%; ertapenem for 66.4%). These results show why carbapenems are used in the treatment of infections by multidrug-resistant bacteria. The rate of imipenem resistance in this study could be due to chromosomal mechanisms by porin alteration, the combination of resistance mechanisms (ΒLSE and/or cephalosporinase associated with loss of membrane permeability) or the production of carbapenemases [28] .
The spread of blaIMP gene-mediated resistance is a concerning issue in healthcare settings and communities worldwide. It can lead to difficult-to-treat infections and can have serious implications for public health, as it may result in the limited effectiveness of available antibiotics [29] . To combat this, it is essential to monitor and track the prevalence of blaIMP gene in bacterial strains to implement appropriate infection control measures and preserve the efficacy of existing antibiotic treatments [30] . In this study, we obtained 4 strains positive (23.53%) for IMP-type metallo-β-lactamases including 3 strains of Klebsiella pneumoniae and 01 strain of Klebsiella oxytoca. The presence of IMP-type metallo-β-lactamase has been reported in Klebsiella pneumoniae in several countries such as Iran [27] [31] , the United States [32] , China [33] [34] , Brazil [35] and Japan [36] . The presence of the IMP-type metallo-β-lactamase has also been detected in China [37] and Australia [38] . The prevalence of the IMP gene has been reported in Gram-negative bacilli in Tanzania [39] , Egypt [40] [41] and Sudan [42] at 12%; 4.2%; 20%; and 26.4%, respectively. All this testifies to the global distribution of IMP-type carbapenemase-producing Enterobacteriaceae and the very high variability of their prevalence depending on the country. The presence of this gene in our study could be explained by the fact that this gene is usually located on mobile genetic elements (plasmids, transposons, integrons) allowing for rapid dissemination worldwide [28] [43] . The presence of this gene would explain the multidrug resistance of strains carrying it. One of the limitations of this study is that it focused on bacterial strains collected between 2009 and 2013. However, it provides an overview of antimicrobial resistance of clinical strains of Klebsiella in Burkina Faso. Research and development of new antimicrobial agents and strategies are crucial in addressing the challenges posed by blaIMP-mediated resistance and other antibiotic resistance mechanisms.
5. Conclusion
Altogether the present study showed high resistance of Klebsiella to third generation cephalosporins and a low resistance rate to imipenem. We were also able to identify the blaIMP gene. Multidrug resistance in Enterobacteriaceae raises public health concerns. Surveillance measures must be taken to prevent the emergence of carbapenemase-producing bacteria through enzyme production.
Acknowledgements
The authors wish to thank all participants in this study. A deep gratitude to all the staff of Saint Camille Hospital of Ouagadougou (HOSCO) and Biomolecular Research Center Pietro Annigoni (CERBA) for technical support.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.
Cite this paper
Ouédraogo, B., Ouattara, A.K., Dabiré, A.M., Tiemtoré, R.Y.W., Sougé, S. and Simporé, J. (2023) Resistance of Klebsiella to Imipenem by Production of Carbapenemase Gene blaIMP at Centre Hospitalier Universitaire Pédiatrique Charles de Gaulle, Ouagadougou, Burkina Faso. International Journal of Clinical Medicine, 14, 347-356. https://doi.org/10.4236/ijcm.2023.148030
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