Evaluation of antimicrobial combination therapy options for the management of integron-mediated multidrug resistance in enterococcus species and acinetobacter baumannii from aquatic environment in the Eastern Cape Province, South Africa
- Authors: Ola, Adeniji Oluwaseun
- Date: 2022-04
- Subjects: Enterococcus , Aquatic biodiversity , Acinetobacter
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27776 , vital:69467
- Description: Infectious diseases caused by multidrug-resistant MDR pathogens, such as Acinetobacter baumannii and Enterococcus spp., is an increasing worldwide problem. For treating these diseases, antibiotics are usually the first choice. But organisms develop resistance as a result of drug abuse, continuous use of antibiotics and release of antibiotics into the environment. These have prompted MDR's development, making even the most active drugs ineffective. Transposons, plasmids and integrons are the most effective mobile genetic elements that promote acquisition and spread of resistance determinants. Integrons carrying various arrays of resistance gene cassettes are principally helpful for epidemiological studies of these disease-causing organisms. Alternative treatments, such as using drugs in combination or with adjuvants and nanoparticles therapy, have been documented. Nanoparticles have the potential requirements for qualifying as antibacterial agents. In addition to their antimicrobial activities, nanoparticles can be used together with antibiotics for more enhanced antimicrobial activity In this study, Acinetobacter baumanni, Enterococcus faecium and Enterococcus faecalis were recovered from the aquatic environment in the Eastern Cape Province, South Africa with a standard microbiological method. Their antibiotic sensitivity testing was carried out using Kirby Bauer disk diffusion and microdilution methods. A high occurrence of class-1 integrons was discovered in MDR A. baumanni, with the internal variable containing aadA1, aadA5 and aadA2 genes, which confer resistance for streptomycin and spectinomycin, aac 6Ib for amikacin/ tobramycin and dfrA17 genes for trimethoprim. Similarly, class1 integron was detected in Enterococcus, without the presence of gene cassette. The checkerboard assay and time-kill assay were used to test for the effect of the combination of the antibiotic. The impact of colistin combined with quinolones (ciprofloxacin) with the Fractional inhibitory concentration index FICs 0.31 indicated synergistic effects against MDR A baumanni. However, when colistin was combined with meropenem and ceftazidime, additive effects with FIC, ranging from 0.52 to 1 were observed. In addition, a combination of gentamicin MIC 4 μgml with vancomycin MIC 256 μgml antibiotics against vancomycin-resistant Enterococcus faecalis showed antibacterial activity. In contrast, the combination of ciprofloxacin 1 μgml with Ampicillin 16 μgml antibiotics against Enterococcus faecalis showed a bacteriostatic effect. The initial inoculum declined by 100 percentage when gentamicin was combined with vancomycin at a concentration of 4 and 128 μgml MIC respectively, for about 2 h following the treatment for MDR E. faecium. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction ERIC PCR analyses of the studied pathogens revealed great genetic diversity, suggesting the various sources of environmental contamination. Silver nanoparticles AgNPs and zinc oxide nanoparticles ZnO NPs were chemically synthesized using a precipitation method and characterized using energy dispersive Xray analysis EDX, scanning electron microscopy SEM, Fourier transforms infrared spectroscopic analysis FTIR and transmission electron microscopy TEM. The characterization results showed the synthesis of 43.37 nm and 21.03 nm nanoparticles of zinc oxide and silver origins, correspondingly, with distinct morphology, as revealed in TEM. The size, stability and functional groups of the nanoparticles produced were revealed using EDX and FTIR. Zinc oxide nanoparticles were cytotoxic against Vero cell lines at the tested concentrations, whereas AgNPs had no cytotoxic effect at lower concentrations. Acinetobacter baumanni, Enterococcus faecium and Enterococcus faecalis were with MIC in the range of 0.0390.078mgml for silver nanoparticles. Zinc oxide nanoparticles were explicitly active against Enterococcus species gram-positive with MIC of 1.25 5 mgml. Both studied nanoparticles exhibited profound synergistic and additive activities against all the investigated MDR pathogens. These findings demonstrate good antibacterial potential of the nanoparticles against drug-resistant strains and open a new arena of antimicrobials for medical treatment. , Thesis (MSc) -- Faculty of Science and Agriculture, 2022
- Full Text:
- Date Issued: 2022-04
- Authors: Ola, Adeniji Oluwaseun
- Date: 2022-04
- Subjects: Enterococcus , Aquatic biodiversity , Acinetobacter
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27776 , vital:69467
- Description: Infectious diseases caused by multidrug-resistant MDR pathogens, such as Acinetobacter baumannii and Enterococcus spp., is an increasing worldwide problem. For treating these diseases, antibiotics are usually the first choice. But organisms develop resistance as a result of drug abuse, continuous use of antibiotics and release of antibiotics into the environment. These have prompted MDR's development, making even the most active drugs ineffective. Transposons, plasmids and integrons are the most effective mobile genetic elements that promote acquisition and spread of resistance determinants. Integrons carrying various arrays of resistance gene cassettes are principally helpful for epidemiological studies of these disease-causing organisms. Alternative treatments, such as using drugs in combination or with adjuvants and nanoparticles therapy, have been documented. Nanoparticles have the potential requirements for qualifying as antibacterial agents. In addition to their antimicrobial activities, nanoparticles can be used together with antibiotics for more enhanced antimicrobial activity In this study, Acinetobacter baumanni, Enterococcus faecium and Enterococcus faecalis were recovered from the aquatic environment in the Eastern Cape Province, South Africa with a standard microbiological method. Their antibiotic sensitivity testing was carried out using Kirby Bauer disk diffusion and microdilution methods. A high occurrence of class-1 integrons was discovered in MDR A. baumanni, with the internal variable containing aadA1, aadA5 and aadA2 genes, which confer resistance for streptomycin and spectinomycin, aac 6Ib for amikacin/ tobramycin and dfrA17 genes for trimethoprim. Similarly, class1 integron was detected in Enterococcus, without the presence of gene cassette. The checkerboard assay and time-kill assay were used to test for the effect of the combination of the antibiotic. The impact of colistin combined with quinolones (ciprofloxacin) with the Fractional inhibitory concentration index FICs 0.31 indicated synergistic effects against MDR A baumanni. However, when colistin was combined with meropenem and ceftazidime, additive effects with FIC, ranging from 0.52 to 1 were observed. In addition, a combination of gentamicin MIC 4 μgml with vancomycin MIC 256 μgml antibiotics against vancomycin-resistant Enterococcus faecalis showed antibacterial activity. In contrast, the combination of ciprofloxacin 1 μgml with Ampicillin 16 μgml antibiotics against Enterococcus faecalis showed a bacteriostatic effect. The initial inoculum declined by 100 percentage when gentamicin was combined with vancomycin at a concentration of 4 and 128 μgml MIC respectively, for about 2 h following the treatment for MDR E. faecium. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction ERIC PCR analyses of the studied pathogens revealed great genetic diversity, suggesting the various sources of environmental contamination. Silver nanoparticles AgNPs and zinc oxide nanoparticles ZnO NPs were chemically synthesized using a precipitation method and characterized using energy dispersive Xray analysis EDX, scanning electron microscopy SEM, Fourier transforms infrared spectroscopic analysis FTIR and transmission electron microscopy TEM. The characterization results showed the synthesis of 43.37 nm and 21.03 nm nanoparticles of zinc oxide and silver origins, correspondingly, with distinct morphology, as revealed in TEM. The size, stability and functional groups of the nanoparticles produced were revealed using EDX and FTIR. Zinc oxide nanoparticles were cytotoxic against Vero cell lines at the tested concentrations, whereas AgNPs had no cytotoxic effect at lower concentrations. Acinetobacter baumanni, Enterococcus faecium and Enterococcus faecalis were with MIC in the range of 0.0390.078mgml for silver nanoparticles. Zinc oxide nanoparticles were explicitly active against Enterococcus species gram-positive with MIC of 1.25 5 mgml. Both studied nanoparticles exhibited profound synergistic and additive activities against all the investigated MDR pathogens. These findings demonstrate good antibacterial potential of the nanoparticles against drug-resistant strains and open a new arena of antimicrobials for medical treatment. , Thesis (MSc) -- Faculty of Science and Agriculture, 2022
- Full Text:
- Date Issued: 2022-04
Surveillance study on pathogenic Acinetobacter species in freshwater environment of the Amathole and Chris Hani District Municipalities, Eastern Cape, South Africa
- Authors: Adewoyin, Mary Ayobami
- Date: 2019-09
- Subjects: Acinetobacter infections , Acinetobacter , Nosocomial infections
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/19836 , vital:43257
- Description: This study evaluates the occurrence of medically relevant Acinetobacter species in three rivers, namely; Keiskamma, Tyhume and Great Fish in the Eastern Cape Province, South Africa in one year sampling regime (April 2017 - March 2018). The physicochemical parameters (pH, temperature (TEM), electrical conductivity (EC), total dissolved solids (TDS), salinity (SAL), total suspended solids (TSS), turbidity (TBS), dissolved oxygen (DO) and biological oxygen demand (BOD)) of the water bodies were measured. The presumptive Acinetobacter species recovered from the freshwater resources were recorded and further confirmed using molecular techniques. Similarly, confirmed isolates were subjected to speciation using species-specific primer sets for A. baumannii and A. nosocomialis. Also, virulence genes namely; afa/draBC, epsA, fimH, OmpA, PAI, sfa/focDE, and traT in the two Acinetobacter species were also determined using molecular method. In addition, the antibiogram characteristics of A. baumannii and A. nosocomialis isolated from the water samples were determined using standard methods. The antibiotic susceptibility test was performed using a panel of 12 antibiotics belonging to the aminoglycosides (amikacin, AK and gentamicin, GM), β-lactam/β-lactamase-inhibitor combinations (piperacillin-tazobactam, PTZ), cephems (ceftazidime, CAZ, cefotaxime, CTX, and cefepime, CPM), carbapenems (imipenem, IMI and meropenem, MEM), fluoroquinolones (ciprofloxacin, CIP), folate pathway inhibitors (Trimethoprim/sulfamethoxazole, TS), lipopeptides (Polymyxin B, PB) and tetracyclines (tetracycline, TET). Similarly, antibiotic resistance genes (ARGs) present in the Acinetobacter isolates were investigated including aminoglycoside resistance genes (aacC2, aphA1 and aphA2), β-lactamases resistance genes (blaTEM, blaSHV, blaOXA-1-like, blaCTX-M(GROUP 1), blaCTX-M(GROUP 2), blaCTX-M(GROUP 9), blaVEB, blaGES, blaPER, blaCTX-M-8/-25, blaOXA-48-like, blaVIM, blaIMP and blaKPC), fluoroquinolones resistance genes (qnrA, qnrB, qnrC, qnrD and qnrS), sulfonamide resistance genes (sul1 and sul2), and tetracycline resistance genes (tetA, tetB, tetC, tetM, tetL and tetO). The pH, EC, TDS, SAL, TEMP, TSS, TBS, DO, and BOD for Tyhume River ranged as follows: 7.2-7.7, 125-141 μS/cm, 62-71mg/L, 0.06- 0.07 PSU, 11.3-20.2oC, 30.0-89.6 mg/L, 35.0-96.0 NTU, 8.2-9.8 mg/L, 2.0-4.2 mg/L, while for Great Fish River, the parameters were 8.0-8.2, 274-369 μS/cm, 137-184mg/L, 0.13-0.18PSU, 12.7-22.3oC, 44.3-99.4 mg/L, 48.0-214.0 NTU, 7.8-9.9 mg/L, 3.1- 4.9 mg/L, and at Keiskamma River they were 7.5-7.9, 153.2-285.0 μS/cm, 86-143 mg/L, 0.07-0.14 PSU, 11.0-21.4oC, 27.0- 55.6 mg/L, 31-61 NTU, 8.3-9.8 mg/L, 3.0-6.0 mg/L. A total of 1107 presumptive Acinetobacter spp. were recovered from the rivers sampled of which 428, 370 and 309 isolates were recovered from Tyhume, Great Fish and Keiskamma rivers respectively. However, only 844 was confirmed positive for the genus Acinetobacter and are recovered in the proportions 285 (77 percent), 219 (70.9 percent) and 340 (79 percent) from Great Fish, Keiskemma and Tyhume rivers respectively. Our finding revealed that 410 (48.58 percent) and 23 (2.7 percent) of the isolates were confirmed to be A. baumannii and A. nosocomalis respectively. Also, 308 (75.12 percent percent) A. baumannii and 3 (13.04 percent) A. nosocomialis isolates exhibited one or more virulence genes out of the seven tested, whereas 102 (24.88 percent) and 20 (86.95 percent) of the A. baumannii and A. nosocomialis isolates did not harbour any virulence gene. Additionally, OmpA was the most prevalent (p<0.05) virulence gene found in A. baumannii with 69 (45.10 percent), 52 (50.98 percent) and 77 (49.68 percent) isolates from Great Fish, Keiskamma and Tyhume rivers respectively. The rates of susceptibilities of A. baumannii and A. nosocomialis to the antibiotics followed the order; Piperacillin-tazobactam (72.8 percent ; 73.9 percent), Ceftazidime (70.5 percent ; 91.3 percent), Cefotaxime (16.8 percent ; 17.4 percent), Cefepime (88.5 percent ; 95.7 percent), Imipenem (95.9 percent ; 100 percent ), Meropenem (92.7 percent ; 91.3 percent), Amikacin (97.6 percent ; 91.3 percent), Gentamicin (89.8 percent ; 87 percent), Polymyxin B (84.4 percent ; 91.3 percent), Tetracycline (74.7 percent ; 78.3 percent), Ciprofloxacin (75.9 percent ; 78.3 percent) and Trimethoprim/sulfamethoxazole (74.0 percent ; 73.9 percent) respectively. Both A. baumannii and A. nosocomialis were highly susceptible to all the antimicrobials tested except cefotaxime where 64 percent and 78 percent intermediate responses were observed in the species. At least 10 isolates of A. baumannii were resistant against each of the antibiotics used. The modal multiple antibiotics resistance phenotypes (MARPs) for Acinetobacter spp. was MARP 3 (29.87 percent) and the least was MARP 10 and 11 (2.6 percent each). The antimicrobial resistance index (ARI) was higher at two sampling sites KE2 (0.33) and TY1 (0.22). Similarly, MARI showed that sampling sites KE2 was a hotspot for multidrug-resistant Acinetobacter spp. Of the five classes of ARGs studied, there was a widespread of β-lactamases (blaTEM) in the two Acinetobacter species, followed by sul2, which were detected in 67 (63.2 percent) and 44 (49.4 percent) isolates respectively, across the rivers studied. We conclude that aquatic resources of the study community are important reservoirs of pathogenic Acinetobacter species and antibiotic resistance determinants. The occurrence of clinically-important Acinetobacter species suggests possible contamination of these selected rivers which are consumed by humans and livestock, as well as being used for irrigation system, and this constitutes a risk to public health. It also shows that A. baumannii and A. nosocomialis can thrive in the aquatic environment. This study suggests that direct utilization of water from these sources for domestic and other purposes without any form of pre-treatment should be avoided. It is, therefore, necessary for regulatory authorities to monitor the release of domestic and industrial wastewater into these water bodies in order to prevent outbreaks of epidemics. , Thesis (PhD) (Microbiology) -- University of Fort Hare, 2019
- Full Text:
- Date Issued: 2019-09
- Authors: Adewoyin, Mary Ayobami
- Date: 2019-09
- Subjects: Acinetobacter infections , Acinetobacter , Nosocomial infections
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/19836 , vital:43257
- Description: This study evaluates the occurrence of medically relevant Acinetobacter species in three rivers, namely; Keiskamma, Tyhume and Great Fish in the Eastern Cape Province, South Africa in one year sampling regime (April 2017 - March 2018). The physicochemical parameters (pH, temperature (TEM), electrical conductivity (EC), total dissolved solids (TDS), salinity (SAL), total suspended solids (TSS), turbidity (TBS), dissolved oxygen (DO) and biological oxygen demand (BOD)) of the water bodies were measured. The presumptive Acinetobacter species recovered from the freshwater resources were recorded and further confirmed using molecular techniques. Similarly, confirmed isolates were subjected to speciation using species-specific primer sets for A. baumannii and A. nosocomialis. Also, virulence genes namely; afa/draBC, epsA, fimH, OmpA, PAI, sfa/focDE, and traT in the two Acinetobacter species were also determined using molecular method. In addition, the antibiogram characteristics of A. baumannii and A. nosocomialis isolated from the water samples were determined using standard methods. The antibiotic susceptibility test was performed using a panel of 12 antibiotics belonging to the aminoglycosides (amikacin, AK and gentamicin, GM), β-lactam/β-lactamase-inhibitor combinations (piperacillin-tazobactam, PTZ), cephems (ceftazidime, CAZ, cefotaxime, CTX, and cefepime, CPM), carbapenems (imipenem, IMI and meropenem, MEM), fluoroquinolones (ciprofloxacin, CIP), folate pathway inhibitors (Trimethoprim/sulfamethoxazole, TS), lipopeptides (Polymyxin B, PB) and tetracyclines (tetracycline, TET). Similarly, antibiotic resistance genes (ARGs) present in the Acinetobacter isolates were investigated including aminoglycoside resistance genes (aacC2, aphA1 and aphA2), β-lactamases resistance genes (blaTEM, blaSHV, blaOXA-1-like, blaCTX-M(GROUP 1), blaCTX-M(GROUP 2), blaCTX-M(GROUP 9), blaVEB, blaGES, blaPER, blaCTX-M-8/-25, blaOXA-48-like, blaVIM, blaIMP and blaKPC), fluoroquinolones resistance genes (qnrA, qnrB, qnrC, qnrD and qnrS), sulfonamide resistance genes (sul1 and sul2), and tetracycline resistance genes (tetA, tetB, tetC, tetM, tetL and tetO). The pH, EC, TDS, SAL, TEMP, TSS, TBS, DO, and BOD for Tyhume River ranged as follows: 7.2-7.7, 125-141 μS/cm, 62-71mg/L, 0.06- 0.07 PSU, 11.3-20.2oC, 30.0-89.6 mg/L, 35.0-96.0 NTU, 8.2-9.8 mg/L, 2.0-4.2 mg/L, while for Great Fish River, the parameters were 8.0-8.2, 274-369 μS/cm, 137-184mg/L, 0.13-0.18PSU, 12.7-22.3oC, 44.3-99.4 mg/L, 48.0-214.0 NTU, 7.8-9.9 mg/L, 3.1- 4.9 mg/L, and at Keiskamma River they were 7.5-7.9, 153.2-285.0 μS/cm, 86-143 mg/L, 0.07-0.14 PSU, 11.0-21.4oC, 27.0- 55.6 mg/L, 31-61 NTU, 8.3-9.8 mg/L, 3.0-6.0 mg/L. A total of 1107 presumptive Acinetobacter spp. were recovered from the rivers sampled of which 428, 370 and 309 isolates were recovered from Tyhume, Great Fish and Keiskamma rivers respectively. However, only 844 was confirmed positive for the genus Acinetobacter and are recovered in the proportions 285 (77 percent), 219 (70.9 percent) and 340 (79 percent) from Great Fish, Keiskemma and Tyhume rivers respectively. Our finding revealed that 410 (48.58 percent) and 23 (2.7 percent) of the isolates were confirmed to be A. baumannii and A. nosocomalis respectively. Also, 308 (75.12 percent percent) A. baumannii and 3 (13.04 percent) A. nosocomialis isolates exhibited one or more virulence genes out of the seven tested, whereas 102 (24.88 percent) and 20 (86.95 percent) of the A. baumannii and A. nosocomialis isolates did not harbour any virulence gene. Additionally, OmpA was the most prevalent (p<0.05) virulence gene found in A. baumannii with 69 (45.10 percent), 52 (50.98 percent) and 77 (49.68 percent) isolates from Great Fish, Keiskamma and Tyhume rivers respectively. The rates of susceptibilities of A. baumannii and A. nosocomialis to the antibiotics followed the order; Piperacillin-tazobactam (72.8 percent ; 73.9 percent), Ceftazidime (70.5 percent ; 91.3 percent), Cefotaxime (16.8 percent ; 17.4 percent), Cefepime (88.5 percent ; 95.7 percent), Imipenem (95.9 percent ; 100 percent ), Meropenem (92.7 percent ; 91.3 percent), Amikacin (97.6 percent ; 91.3 percent), Gentamicin (89.8 percent ; 87 percent), Polymyxin B (84.4 percent ; 91.3 percent), Tetracycline (74.7 percent ; 78.3 percent), Ciprofloxacin (75.9 percent ; 78.3 percent) and Trimethoprim/sulfamethoxazole (74.0 percent ; 73.9 percent) respectively. Both A. baumannii and A. nosocomialis were highly susceptible to all the antimicrobials tested except cefotaxime where 64 percent and 78 percent intermediate responses were observed in the species. At least 10 isolates of A. baumannii were resistant against each of the antibiotics used. The modal multiple antibiotics resistance phenotypes (MARPs) for Acinetobacter spp. was MARP 3 (29.87 percent) and the least was MARP 10 and 11 (2.6 percent each). The antimicrobial resistance index (ARI) was higher at two sampling sites KE2 (0.33) and TY1 (0.22). Similarly, MARI showed that sampling sites KE2 was a hotspot for multidrug-resistant Acinetobacter spp. Of the five classes of ARGs studied, there was a widespread of β-lactamases (blaTEM) in the two Acinetobacter species, followed by sul2, which were detected in 67 (63.2 percent) and 44 (49.4 percent) isolates respectively, across the rivers studied. We conclude that aquatic resources of the study community are important reservoirs of pathogenic Acinetobacter species and antibiotic resistance determinants. The occurrence of clinically-important Acinetobacter species suggests possible contamination of these selected rivers which are consumed by humans and livestock, as well as being used for irrigation system, and this constitutes a risk to public health. It also shows that A. baumannii and A. nosocomialis can thrive in the aquatic environment. This study suggests that direct utilization of water from these sources for domestic and other purposes without any form of pre-treatment should be avoided. It is, therefore, necessary for regulatory authorities to monitor the release of domestic and industrial wastewater into these water bodies in order to prevent outbreaks of epidemics. , Thesis (PhD) (Microbiology) -- University of Fort Hare, 2019
- Full Text:
- Date Issued: 2019-09
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