- Title
- Molecular characterization of integrons and their associated gene cassettes in multidrug-resistant enterobacteriaceae isolates from environmental sources and the exploration of antibiotic combination against some resistant strains
- Creator
- Fadare, Folake Temitope https://orcid.org/0000-0001-5779-9798
- Subject
- Enterobacteriaceae
- Subject
- Molecular microbiology
- Subject
- Enterobacteria
- Date
- 2023
- Type
- Doctoral theses
- Type
- text
- Identifier
- http://hdl.handle.net/10353/27809
- Identifier
- vital:69942
- Description
- Globally, the increasing rate of antimicrobial resistance against our currently available drugs has been a serious public concern. Due to the selective nature of antibiotics, bacteria are expected to develop resistance against them over time, but the current scourge of antimicrobial resistance is aggravated by factors other than the expected evolutionary trend. The use and overuse of antibiotics in clinical and agricultural contexts have led to the fast rise of multidrug-resistant MDR microorganisms. A scenario that necessitates an upsurge in the clinical failures observed with our current drug arsenals is expected to rise if left unchecked. One of the significant drivers implicated in the spread of antimicrobial resistance genes is the integrons. These are mobile genetic elements found on pathogenicity islands, transposons, and plasmids, easing their distribution among various bacteria. They are considered efficient gene expression systems that naturally capture, integrate gene cassettes GCs and immediately express the captured antimicrobial resistance genes on the GCs due to the inherent promoters on their structures. Integrons have been known to confer resistance against most classes of antibiotics. These include all known β-lactams, chloramphenicol, trimethoprim, erythromycin, aminoglycosides, quinolones, streptothricin, lincomycin, rifampicin, fosfomycin, and antiseptics of the quaternary ammonium compound family. They have been detected in bacterial populations under direct or indirect antibiotic pressure in clinical, agricultural, and environmental contexts. The emergence of MDR in Enterobacteriaceae is a critical public health issue that has attracted the World Health Organization WHO, which classified them as one of the critical priority pathogens urgently requiring new antibiotics. The resistance phenomenon has proven most of the current antibiotics ineffective, further compounded by the slow pace of the discovery of new antibiotics, necessitating the hunt for new, practical remedies. One of such is the exploration of synergy among existing antibiotics. Two medications combined have a higher impact, thereby allowing current antibiotics to be salvaged for use in treating MDR bacteria, even if the bacteria are resistant against one or both antibiotics separately. Hence, this research focused on the occurrence and prevalence of multidrug resistance and the characterization of integrons and their associated gene cassettes in members of Enterobacteriaceae, including Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Escherichia coli, and Citrobacter spp. recovered from animal droppings, rivers, and effluents of hospital and wastewater treatment plants in Eastern Cape Province, South Africa. The inhibitory effect of combining two drugs belonging to different antibiotic classes to obtain a possible potentiating effect against some multidrug-resistant Enterobacteriaceae isolates harbouring integrons were examined and studied. The isolates were identified using the conventional molecular Polymerase Chain Reaction with specific primers. The antimicrobial resistance profile and the production of Extended-spectrum and metallo β-lactamase were detected using disk diffusion technique DDT, double-disk synergy test DDST, and ethylenediaminetetraacetic acid EDTA tests, respectively. The PCR-based screening method, DNA sequencing analyses, and restriction fragment length polymorphism RFLP were used to characterize the integrons and their associated GCs. Furthermore, Enterobacterial Repetitive Intergenic Consensus ERIC PCR determined the genotypic relationships between some specific species. The various antibiotics' minimum inhibitory concentration MIC was determined using the broth microdilution, while the checkerboard method was used to determine the fractional inhibitory concentration indices FICIs. The time-kill assays TKAs were further used to confirm the synergism observed from the checkerboard assays. Most of the isolates were resistant against most antibiotics tested and were considered MDR. The least resistance was observed against imipenem, a carbapenem, one of the drugs of last resort. Also present were the ESBL and MBL producers, with a few isolates co-producing the enzymes. A high prevalence of integrons was observed in the isolates, with class 1 integrons being the most frequently detected. Some isolates co-harboured the intI1 and intI2 genes and were classified as class1 plus 2 integrons. Although Citrobacter spp. had the least number of isolates among the Enterobacteriaceae studied, it harboured the most diverse gene cassette arrays. The various gene cassette arrays were identified as follows: For Klebsiella spp. Aac 6 Ib, aadA1 dfrA1, and dfrA1 sat2; for Citrobacter spp., dfrA5 aac3 Ib, aac6 ib, aadA1dfrA1 aadA1, aadA1-dfrA1, aadA5 dfrA17, and dfrA21-aac3-Ib; for E. coli dfrA21- aac-3-Ib, dfrA5-aac-3-Ib, aadA1 dfrA1, and aadA5 dfrA17 and for E. cloacae aadA1 dfrA1, dfrA7 dfrA21 dfrA5 aac 3 Ib, and dfrA1 sat2. The GC array dfrA1 sat2 was the only array detected in class 2 integrons which are analogous to that found in Tn7, dfrA1-sat2-aadA1, with the deletion of the last GC aadA1. These detected GCs confer resistance against aminoglycosides, including streptomycin and spectinomycin, and trimethoprim, further increasing the resistance spectrum of the bacterial species harbouring them. The detection of integrons and their associated GC and the presence of these β-lactamases is also associated with coresistance against other classes of antibiotics by bacterial species harbouring them, further limiting treatment options. The checkerboard assays combining antibiotics against these drug-resistant integron harbouring Enterobacteriaceae revealed that 26.3 percent 10 over 38 of the interactions were categorized as synergistic, while 73.7 percent 28 over 38 were indifferent. None of the combinations was antagonistic. The TKAs revealed all the synergistic interactions as bactericidal. Therefore, the combinations of gentamicin with tetracycline, ciprofloxacin, and ceftazidime against Multidrug-resistant MDR Klebsiella pneumoniae; tetracycline-ceftazidime combination against MDR Escherichia coli, colistin combinations with ceftazidime and gentamicin, and tetracycline-gentamicin combinations against MDR Citrobacter freundii may be future therapeutic alternatives.
- Description
- Thesis (MSci) -- Faculty of Science and Agriculture, 2023
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (xxiii, 302 leaves)
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
- Rights
- All Rights Reserved
- Rights
- Open Access
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UFH Department of Biochemistry & Microbiology
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details | SOURCE1 | Fadare_FT_Thesis - FINAL.pdf | 4 MB | Adobe Acrobat PDF | View Details |