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
- Fadare, Folake Temitope https://orcid.org/0000-0001-5779-9798
- Authors: Fadare, Folake Temitope https://orcid.org/0000-0001-5779-9798
- Date: 2023
- Subjects: Enterobacteriaceae , Molecular microbiology , Enterobacteria
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27809 , 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. , Thesis (MSci) -- Faculty of Science and Agriculture, 2023
- Full Text:
- Authors: Fadare, Folake Temitope https://orcid.org/0000-0001-5779-9798
- Date: 2023
- Subjects: Enterobacteriaceae , Molecular microbiology , Enterobacteria
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27809 , 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. , Thesis (MSci) -- Faculty of Science and Agriculture, 2023
- Full Text:
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:
- 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
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Genetic diversity, resistance profile of HIV and risk assessment of mother-to-child transmission in pregnant women on anti-retroviral therapy in the Eastern Cape, South Africa
- Authors: Adeniyi, Oladele Vincent
- Date: 2018-12
- Subjects: Antiretroviral agents , AIDS (Disease) in infants
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/19817 , vital:43254
- Description: Despite the initiation of life-long ART in HIV-infected pregnant women, the rate and determinants of infant HIV transmission are not known, especially in the poor resource settings of the Eastern Cape, South Africa. Maternal anti-retroviral therapy (ART) is crucial for elimination of mother-to-child transmission (MTCT) of HIV. However, the inevitable risks of emergence of HIV drug resistance poses significant threat to achieving this goal of HIV-free generation and keeping mothers alive. Also, it is unclear if women with high viral load at delivery have acquired clinically relevant mutations, which could confer resistance to the ART, thus, further increasing the risks of motherto- child transmission of HIV-drug resistance strains. In addition to the gaps identified in the prevention of mother-to-child transmission (PMTCT) context, the understanding of regional epidemics is crucial to the broader epidemiological profiling of HIV infections in the country. Despite the rapid influx of foreign nationals to South African and Eastern Cape Province, there has not been any molecular epidemiological studies profiling the HIV diversity in the Eastern Cape. , Thesis (PhD) (Microbiology) -- University of Fort Hare, 2018
- Full Text:
- Authors: Adeniyi, Oladele Vincent
- Date: 2018-12
- Subjects: Antiretroviral agents , AIDS (Disease) in infants
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/19817 , vital:43254
- Description: Despite the initiation of life-long ART in HIV-infected pregnant women, the rate and determinants of infant HIV transmission are not known, especially in the poor resource settings of the Eastern Cape, South Africa. Maternal anti-retroviral therapy (ART) is crucial for elimination of mother-to-child transmission (MTCT) of HIV. However, the inevitable risks of emergence of HIV drug resistance poses significant threat to achieving this goal of HIV-free generation and keeping mothers alive. Also, it is unclear if women with high viral load at delivery have acquired clinically relevant mutations, which could confer resistance to the ART, thus, further increasing the risks of motherto- child transmission of HIV-drug resistance strains. In addition to the gaps identified in the prevention of mother-to-child transmission (PMTCT) context, the understanding of regional epidemics is crucial to the broader epidemiological profiling of HIV infections in the country. Despite the rapid influx of foreign nationals to South African and Eastern Cape Province, there has not been any molecular epidemiological studies profiling the HIV diversity in the Eastern Cape. , Thesis (PhD) (Microbiology) -- University of Fort Hare, 2018
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Distribution of Escherichia coli O157:H7 in irrigation water, drinking water troughs, dairy wastewater and rectal swabs from three dairy farms in Amathole district municipality, Eastern Cape, South Africa
- Myataza, Asive https://orcid.org/0000-0001-5483-122X
- Authors: Myataza, Asive https://orcid.org/0000-0001-5483-122X
- Date: 2016
- Subjects: Escherichia coli O157:H7 , Irrigation water
- Language: English
- Type: Master's theses
- Identifier: http://hdl.handle.net/10353/24150 , vital:62397
- Description: Escherichia coli belongs to the genus Escherichia which has five species, including E. hermanii, E. fergusonii, E. vulneris, E. blattae and E. coli (Willshaw et al., 2001). Some E. coli strains are pathogenic, and such strains are differentiated into different pathotypes based on the virulence factors they possess, mechanisms of pathogenesis, clinical manifestation and serotyping of their somatic, flagella and capsule (O, H and K) antigens; while some are nonpathogenic (Dikobe et al., 2011). These strains have been grouped into major pathotypes namely: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis E.coli (NMEC) (Willshaw et al., 2001; Villegas-sepulveda et al., 2003; Kaper et al., 2004; Bugarel et al., 2011). Members of EPEC pathotype are Shiga-toxin producing (STEC) organisms, with E. coli O157:H7 serotype being recognized as the most frequent STEC serotype linked to diseases in human (Barkocy-Gallagher et al., 2001; Coombes et al., 2011; Dikobe et al., 2011; Jacob et al., 2013). E. coli O157:H7 is the major cause of haemorrhagic diarrhoea and haemolytic uremic syndrome (UHS) in humans, due to the production of Shiga-toxins that are similar to Shigella toxins (Villegas-sepulveda et al., 2003; Ngwa et al., 2013). These toxins are coded by genes which allow them to penetrate small intestines of human and animals (Chern et al., 2011). Dairy products and cattle are the major reservoirs of haemorrhagic E. coli O157: H7 (Elder et al., 2000; Elmali et al., 2005; Ayaz et al., 2014). Haemorrhagic E. coli O157:H7 is carried mostly in the gastrointestinal tracts of cows and other ruminants (Gun et al., 2003)and it is transmitted through ingestion of contaminated food and water, or through direct and indirect contact with infected cows and other ruminants and/or via a person to person transmission (Lahti et al., 2003). Almost 70percent of countries in the world use poorly treated water for irrigation; this is inclusive of developing countries, which lack well developed means of adequate water treatment due to unavailable financial resources (Gerba et al., 2011). Most communities in the Eastern Cape Province of South Africa are rural with poor water supply with majority of them relying on ground and surface waters that are impacted by insufficiently treated municipal water (which may harbour many pathogens) for domestic purposes (Ateba et al., 2008). Cows may contaminate drinking, recreational and irrigation waters with faecal matters or through direct contact with water (Solomon et al., 2002; Chern et al., 2011). A number of countries with water shortage tend to use insufficiently treated water for irrigation, leading to further contamination of irrigated crops (Fatoki et al., 2001). This has all resulted to elevated levels of E. coli O157:H7 related disease outbreaks and deaths of humans (Olsen et al., 2002). , Thesis (MSc) -- Faculty of Science and Agriculture, 2016
- Full Text:
- Authors: Myataza, Asive https://orcid.org/0000-0001-5483-122X
- Date: 2016
- Subjects: Escherichia coli O157:H7 , Irrigation water
- Language: English
- Type: Master's theses
- Identifier: http://hdl.handle.net/10353/24150 , vital:62397
- Description: Escherichia coli belongs to the genus Escherichia which has five species, including E. hermanii, E. fergusonii, E. vulneris, E. blattae and E. coli (Willshaw et al., 2001). Some E. coli strains are pathogenic, and such strains are differentiated into different pathotypes based on the virulence factors they possess, mechanisms of pathogenesis, clinical manifestation and serotyping of their somatic, flagella and capsule (O, H and K) antigens; while some are nonpathogenic (Dikobe et al., 2011). These strains have been grouped into major pathotypes namely: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis E.coli (NMEC) (Willshaw et al., 2001; Villegas-sepulveda et al., 2003; Kaper et al., 2004; Bugarel et al., 2011). Members of EPEC pathotype are Shiga-toxin producing (STEC) organisms, with E. coli O157:H7 serotype being recognized as the most frequent STEC serotype linked to diseases in human (Barkocy-Gallagher et al., 2001; Coombes et al., 2011; Dikobe et al., 2011; Jacob et al., 2013). E. coli O157:H7 is the major cause of haemorrhagic diarrhoea and haemolytic uremic syndrome (UHS) in humans, due to the production of Shiga-toxins that are similar to Shigella toxins (Villegas-sepulveda et al., 2003; Ngwa et al., 2013). These toxins are coded by genes which allow them to penetrate small intestines of human and animals (Chern et al., 2011). Dairy products and cattle are the major reservoirs of haemorrhagic E. coli O157: H7 (Elder et al., 2000; Elmali et al., 2005; Ayaz et al., 2014). Haemorrhagic E. coli O157:H7 is carried mostly in the gastrointestinal tracts of cows and other ruminants (Gun et al., 2003)and it is transmitted through ingestion of contaminated food and water, or through direct and indirect contact with infected cows and other ruminants and/or via a person to person transmission (Lahti et al., 2003). Almost 70percent of countries in the world use poorly treated water for irrigation; this is inclusive of developing countries, which lack well developed means of adequate water treatment due to unavailable financial resources (Gerba et al., 2011). Most communities in the Eastern Cape Province of South Africa are rural with poor water supply with majority of them relying on ground and surface waters that are impacted by insufficiently treated municipal water (which may harbour many pathogens) for domestic purposes (Ateba et al., 2008). Cows may contaminate drinking, recreational and irrigation waters with faecal matters or through direct contact with water (Solomon et al., 2002; Chern et al., 2011). A number of countries with water shortage tend to use insufficiently treated water for irrigation, leading to further contamination of irrigated crops (Fatoki et al., 2001). This has all resulted to elevated levels of E. coli O157:H7 related disease outbreaks and deaths of humans (Olsen et al., 2002). , Thesis (MSc) -- Faculty of Science and Agriculture, 2016
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Assessment of the water quality, incidence of enteric viruses and microbial risk in the Buffalo River in the Eastern Cape Province of South Africa
- Chigor, Vincent Nnamdigadi https://orcid.org/0000-0002-0811-4526
- Authors: Chigor, Vincent Nnamdigadi https://orcid.org/0000-0002-0811-4526
- Date: 2013-03
- Subjects: Water quality , Water -- Microbiology
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24272 , vital:62596
- Description: Buffalo River is an important water resource in the Eastern Cape Province of South Africa. Over a 1-year period (August 2010–July 2011), the water quality and incidence of human enteric viruses (HEntVs) was assessed, using standard methods and molecular techniques and a total 72 composite water samples collected monthly from a total of 6 sites located on the river and three dams along its course. The sites were selected based on a number of factors including geographical location, anthropogenic activity/major water use, rural/urban status and access. A total of 13 physicochemical parameters were determined using the standard methods. The counts of faecal indicator bacteria (FIB) including total coliforms (TC), faecal coliforms (FC) and enterococci (ENT) were determined by the membrane filtration technique. HEntVs were concentrated using an adsorption-elution method based on cation (Al3+)-coated membrane filter. Real-time quantitative polymerase chain reaction (qPCR) was used for the detection and quantification of human adenoviruses (HAdV), and real-time reverse transcriptase-PCR (RT-qPCR) was used for the quantitative detection of hepatitis A virus (HAV), human rotaviruses (RoV) and enteroviruses (EnV). The detected HAdV were characterized by multiplex conventional/semi-nested PCR methods. The risks for human health constituted by exposure to the detected HEntVs at the six sites were evaluated by a static quantitative microbial risk assessment (QMRA) using both the exponential and beta-Poisson models. Water temperature ranged from 11 to 28oC, while pH varied from 6.6 to 10.7, and turbidity from 1.7 to 133 NTU. Electrical conductivity (EC), total dissolved solids (TDS) and salinity showed drastic variations (42.3-46693 μS/cm, 20.3–23350 mg/L and 0.02–33.8 PSU respectively). The concentrations of other parameters ranged as follows: chloride (3.7–168 mg/L); DO (6.9–11.1); BOD (0.6–9.4); COD (3.7–45.9); nitrite-nitrogen (0.02–0.21); nitrate-nitrogen (1–4.47); and orthophosphate (0.01–1.72). TC, FC and ENT counts were high and ranged from 1.9 × 102–3.8 × 107 cfu/100 mL, 0–3.0×105 cfu/100 mL and 0–5.3 × 105 cfu/100 mL for TC, FC and ENT respectively. Significantly (P<0.05) higher concentrations of FC and ENT were observed at the sampling sites located at the lower reaches of the river compared to the upper reaches. The FIB counts mostly exceeded the maximum limits recommended by national and international guidelines for safe fresh produce irrigation, domestic applications, full-contact recreation and livestock watering. Significant (P<0.01) positive correlations existed between TDS and salinity (r=0.921), between turbidity and each of TC (r=0.552) and FC (r=0.425), as well as between BOD and each of TC (r=0.282), FC (r=0.472) and ENT (r=0.552). Phosphate correlated positively with FC (r=0.424), and nitrate also with the same, FC (r=0.460). A strong positive correlation existed between FC and ENT (r=0.915). There existed a significant (P˂0.01) inverse correlation between enteric viruses and each of water temperature (r=-0.191) and pH (r=-0.234). No correlation could be deduced between enteric viruses and all the tested chemical and bacteriological parameters. HAV, HAdV, RoV and EnV were detected in 43.1percent, 34.7percent, 13.9percent and 9.7percent respectively of the total 72 water samples tested. Two or more viruses were detected in 22.2 percent of the samples. HAdV were detected at 5 of the 6 sampling sites with concentrations ranging from 1.2×101 genome copies (GC)/litre to 4.71×103 GC/litre. Epidemiologically important serotypes, Ad40/41 constituted 83.3percent, while Ad21 made up 16.7percent of all the HAdV detected. HAV was detected at all the sites in significantly (p < 0.05) varying concentrations that ranged from 1.5 × 101–1.9 × 105 GC/litre compared to RoV and EnV. Neither of RoV nor EnV was detected at any of the dams. The detected concentrations at the non-dam sites ranged from 2.5 × 101–2.1 × 103 GC/litre and 1.3 × 101–8.6 × 101 GC/litre for RoV and EnV respectively. The values for the estimated daily risks of enteric virus infection varied with sites and exposure scenario, and ranged from 7.31×10-3–1 (for HAdV), 4.23×10-2–6.54×10-1 (RoV), 2.32×10-4–1.73×10-1 (HAV) and 1.32×10-4–5.70×10-2 (EnV). The yearly risks of infection in individuals exposed to the river/dam water via drinking, recreational, domestic or irrigational activities were unacceptably high, exceeding the acceptable yearly risk of 0.01percent (10-4 infection/person/year) recommended by the USEPA for drinking water. The risks of illness and of death from infection ranged from 6.58×10-5–5.0×10-1 and 6.58×10-9–5.0×10-5 respectively. Data on the physicochemical and bacteriological parameters showed that the Buffalo River water quality was poor, and deteriorated in the plains compared to the upper reaches. These water quality data, the presence of enteric viruses and the QMRA data, that revealed unacceptably high risks of enteric virus infections, and of illness and mortality from the infections, show that the Buffalo River and its dams are contaminated waters that constitute significant public health hazards. Provision of adequate sanitary infrastructure will help prevent source water contamination, and public health education aimed at improving personal, household and community hygiene is imperative. Monitoring enteric viruses in rivers and source water dams is necessary and useful as a risk assessment tool for the exposed population. Future research should consider a comprehensive characterization of the detected viruses. This work is both a significant contribution to the molecular epidemiology of enteric viruses and the first report on molecular detection and quantification of enteric viruses in surface waters in the Eastern Cape. , Thesis (PhD) -- Faculty of Science and Agriculture, 2013
- Full Text:
- Authors: Chigor, Vincent Nnamdigadi https://orcid.org/0000-0002-0811-4526
- Date: 2013-03
- Subjects: Water quality , Water -- Microbiology
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24272 , vital:62596
- Description: Buffalo River is an important water resource in the Eastern Cape Province of South Africa. Over a 1-year period (August 2010–July 2011), the water quality and incidence of human enteric viruses (HEntVs) was assessed, using standard methods and molecular techniques and a total 72 composite water samples collected monthly from a total of 6 sites located on the river and three dams along its course. The sites were selected based on a number of factors including geographical location, anthropogenic activity/major water use, rural/urban status and access. A total of 13 physicochemical parameters were determined using the standard methods. The counts of faecal indicator bacteria (FIB) including total coliforms (TC), faecal coliforms (FC) and enterococci (ENT) were determined by the membrane filtration technique. HEntVs were concentrated using an adsorption-elution method based on cation (Al3+)-coated membrane filter. Real-time quantitative polymerase chain reaction (qPCR) was used for the detection and quantification of human adenoviruses (HAdV), and real-time reverse transcriptase-PCR (RT-qPCR) was used for the quantitative detection of hepatitis A virus (HAV), human rotaviruses (RoV) and enteroviruses (EnV). The detected HAdV were characterized by multiplex conventional/semi-nested PCR methods. The risks for human health constituted by exposure to the detected HEntVs at the six sites were evaluated by a static quantitative microbial risk assessment (QMRA) using both the exponential and beta-Poisson models. Water temperature ranged from 11 to 28oC, while pH varied from 6.6 to 10.7, and turbidity from 1.7 to 133 NTU. Electrical conductivity (EC), total dissolved solids (TDS) and salinity showed drastic variations (42.3-46693 μS/cm, 20.3–23350 mg/L and 0.02–33.8 PSU respectively). The concentrations of other parameters ranged as follows: chloride (3.7–168 mg/L); DO (6.9–11.1); BOD (0.6–9.4); COD (3.7–45.9); nitrite-nitrogen (0.02–0.21); nitrate-nitrogen (1–4.47); and orthophosphate (0.01–1.72). TC, FC and ENT counts were high and ranged from 1.9 × 102–3.8 × 107 cfu/100 mL, 0–3.0×105 cfu/100 mL and 0–5.3 × 105 cfu/100 mL for TC, FC and ENT respectively. Significantly (P<0.05) higher concentrations of FC and ENT were observed at the sampling sites located at the lower reaches of the river compared to the upper reaches. The FIB counts mostly exceeded the maximum limits recommended by national and international guidelines for safe fresh produce irrigation, domestic applications, full-contact recreation and livestock watering. Significant (P<0.01) positive correlations existed between TDS and salinity (r=0.921), between turbidity and each of TC (r=0.552) and FC (r=0.425), as well as between BOD and each of TC (r=0.282), FC (r=0.472) and ENT (r=0.552). Phosphate correlated positively with FC (r=0.424), and nitrate also with the same, FC (r=0.460). A strong positive correlation existed between FC and ENT (r=0.915). There existed a significant (P˂0.01) inverse correlation between enteric viruses and each of water temperature (r=-0.191) and pH (r=-0.234). No correlation could be deduced between enteric viruses and all the tested chemical and bacteriological parameters. HAV, HAdV, RoV and EnV were detected in 43.1percent, 34.7percent, 13.9percent and 9.7percent respectively of the total 72 water samples tested. Two or more viruses were detected in 22.2 percent of the samples. HAdV were detected at 5 of the 6 sampling sites with concentrations ranging from 1.2×101 genome copies (GC)/litre to 4.71×103 GC/litre. Epidemiologically important serotypes, Ad40/41 constituted 83.3percent, while Ad21 made up 16.7percent of all the HAdV detected. HAV was detected at all the sites in significantly (p < 0.05) varying concentrations that ranged from 1.5 × 101–1.9 × 105 GC/litre compared to RoV and EnV. Neither of RoV nor EnV was detected at any of the dams. The detected concentrations at the non-dam sites ranged from 2.5 × 101–2.1 × 103 GC/litre and 1.3 × 101–8.6 × 101 GC/litre for RoV and EnV respectively. The values for the estimated daily risks of enteric virus infection varied with sites and exposure scenario, and ranged from 7.31×10-3–1 (for HAdV), 4.23×10-2–6.54×10-1 (RoV), 2.32×10-4–1.73×10-1 (HAV) and 1.32×10-4–5.70×10-2 (EnV). The yearly risks of infection in individuals exposed to the river/dam water via drinking, recreational, domestic or irrigational activities were unacceptably high, exceeding the acceptable yearly risk of 0.01percent (10-4 infection/person/year) recommended by the USEPA for drinking water. The risks of illness and of death from infection ranged from 6.58×10-5–5.0×10-1 and 6.58×10-9–5.0×10-5 respectively. Data on the physicochemical and bacteriological parameters showed that the Buffalo River water quality was poor, and deteriorated in the plains compared to the upper reaches. These water quality data, the presence of enteric viruses and the QMRA data, that revealed unacceptably high risks of enteric virus infections, and of illness and mortality from the infections, show that the Buffalo River and its dams are contaminated waters that constitute significant public health hazards. Provision of adequate sanitary infrastructure will help prevent source water contamination, and public health education aimed at improving personal, household and community hygiene is imperative. Monitoring enteric viruses in rivers and source water dams is necessary and useful as a risk assessment tool for the exposed population. Future research should consider a comprehensive characterization of the detected viruses. This work is both a significant contribution to the molecular epidemiology of enteric viruses and the first report on molecular detection and quantification of enteric viruses in surface waters in the Eastern Cape. , Thesis (PhD) -- Faculty of Science and Agriculture, 2013
- Full Text:
Production and biochemical characterization of new bioflocculants from bacteria isolated from freshwater and marine environments of the Eastern Cape in South Africa
- Mabinya, Leonard Vuyani https://orcid.org/0000-0002-0682-7282
- Authors: Mabinya, Leonard Vuyani https://orcid.org/0000-0002-0682-7282
- Date: 2013-01
- Subjects: Flocculation , Bacteria
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24228 , vital:62445
- Description: The production and characterization of bioflocculants produced by three bacteria belonging to Arthrobacter, Halomonas and Micrococcus genera and isolated from freshwater and marine environments were evaluated both as axenic cultures and as consortia. The influences of cultutre conditions such as carbon, nitrogen and metal ions sources, as well as initial pH on bioflocculant production by individual isolates were investigated. Both Arthrobacter sp. Raats and Halomonas sp. Okoh utilized urea as a nitrogen source of choice for optimal production of the bioflocculants with Micrococcus sp. Leo having a preference for peptone. All three strains differed in as far as the carbon source of choice was concerned with lactose, glucose and sucrose the preferred carbon sources respectively. Also, all three bacterial strains produced an extracellular bioflocculant aerobically but an intial pH 7.0 of the culture media was suitable for both Arthrobacter sp. Raats and Halomonas sp. Okoh with a slightly alkaline pH of 9.0 preferred by Micrococcus sp. Leo. The presence of Mg2+ cations stimulated bioflocculant production by both Arthrobacter sp. Raats and Micrococcus sp. Leo while Ca2+ resulted in more efficient bioflocculant production by Halomonas sp. Okoh. Chemical analyses revealed the bioflocculants produced by both Halomonas sp. Okoh and Micrococcus sp. Leo to be predominantly polysaccharides whereas Arthrobacter sp. Raats produced principally a glycoprotein composed of about 56percent protein and 25percent total carbohydrate. Response surface methodology (RSM) was used to optimize production medium for bioflocculant production by a consortium of Halomonas sp. Okoh and Micrococcus sp. Leo. Plackett-Burman experimental design showed that fructose, ammonium sulphate and MgCl2 were significant in the high yield of the bioflocculant. Furthermore, central composite design showed that optimal concentration of these critical nutritional sources were 16.14 g/L, 1.55 g/L and 1.88 g/L for fructose, ammonium sulphate and MgCl2 respectively. Quantification of the bioflocculant showed a yield of 6.43 g/L which was in close accord with the predicted value of 6.51 g/L. FTIR spectrometry of the bioflocculant indicated the presence of carboxyl, hydroxyl and amino groups, typical for heteropolysaccharide, while SEM imaging revealed a lattice-like structure. The efficiency of the nutrient optimization suggests suitability for industrial applicability. , Thesis (PhD) -- Faculty of Science and Agriculture, 2013
- Full Text:
- Authors: Mabinya, Leonard Vuyani https://orcid.org/0000-0002-0682-7282
- Date: 2013-01
- Subjects: Flocculation , Bacteria
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24228 , vital:62445
- Description: The production and characterization of bioflocculants produced by three bacteria belonging to Arthrobacter, Halomonas and Micrococcus genera and isolated from freshwater and marine environments were evaluated both as axenic cultures and as consortia. The influences of cultutre conditions such as carbon, nitrogen and metal ions sources, as well as initial pH on bioflocculant production by individual isolates were investigated. Both Arthrobacter sp. Raats and Halomonas sp. Okoh utilized urea as a nitrogen source of choice for optimal production of the bioflocculants with Micrococcus sp. Leo having a preference for peptone. All three strains differed in as far as the carbon source of choice was concerned with lactose, glucose and sucrose the preferred carbon sources respectively. Also, all three bacterial strains produced an extracellular bioflocculant aerobically but an intial pH 7.0 of the culture media was suitable for both Arthrobacter sp. Raats and Halomonas sp. Okoh with a slightly alkaline pH of 9.0 preferred by Micrococcus sp. Leo. The presence of Mg2+ cations stimulated bioflocculant production by both Arthrobacter sp. Raats and Micrococcus sp. Leo while Ca2+ resulted in more efficient bioflocculant production by Halomonas sp. Okoh. Chemical analyses revealed the bioflocculants produced by both Halomonas sp. Okoh and Micrococcus sp. Leo to be predominantly polysaccharides whereas Arthrobacter sp. Raats produced principally a glycoprotein composed of about 56percent protein and 25percent total carbohydrate. Response surface methodology (RSM) was used to optimize production medium for bioflocculant production by a consortium of Halomonas sp. Okoh and Micrococcus sp. Leo. Plackett-Burman experimental design showed that fructose, ammonium sulphate and MgCl2 were significant in the high yield of the bioflocculant. Furthermore, central composite design showed that optimal concentration of these critical nutritional sources were 16.14 g/L, 1.55 g/L and 1.88 g/L for fructose, ammonium sulphate and MgCl2 respectively. Quantification of the bioflocculant showed a yield of 6.43 g/L which was in close accord with the predicted value of 6.51 g/L. FTIR spectrometry of the bioflocculant indicated the presence of carboxyl, hydroxyl and amino groups, typical for heteropolysaccharide, while SEM imaging revealed a lattice-like structure. The efficiency of the nutrient optimization suggests suitability for industrial applicability. , Thesis (PhD) -- Faculty of Science and Agriculture, 2013
- Full Text:
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