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:
- Date Issued: 2023
- 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:
- Date Issued: 2023
Evaluation of an NADPH-dependent assay for inhibition screening of Salmonella enterica DOXP Reguctoisomerase for identification of novel drug hit compounds
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Date Issued: 2020
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Date Issued: 2020
Incidence and antibiogram fingerprints of members of the Enterobacteriaceae family recovered from river water, hospital effluents and vegetables in Chris Hani and Amathole District Municipalities in the Eastern Cape Province
- Authors: Mpaka, Lindelwa
- Date: 2018
- Subjects: Enterobacteriaceae
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/14549 , vital:40003
- Description: The worldwide problem of antimicrobial resistance has limited the spectrum of the current affordable and effective antimicrobials. Infections associated with resistant microorganisms impose a major threat to public health and economic stability. Globally, about 700 000 deaths every year can be accredited to antimicrobial resistance. The leading mechanism of resistance amid bacterial pathogens is the extended spectrum beta-lactamases production, which inhibits spectrum activity of several antimicrobial agents. The rise in antimicrobial resistance has compelled an urgent need of developing means of combatting resistance issue amid diseasecausing microbes. The main aim of this study is to evaluate the incidence and antibiogram fingerprints of Enterobacteriaceae recovered from hospital effluents, river water and vegetables in the Eastern Cape Province. A total of eighteen antibiotics from ten different antimicrobial classes were used to determine antibiogram profiles of the MALDI-TOF confirmed isolates. From the MALDI-TOF confirmed isolates, 60% of Enterobacter spp. and E. coli isolates displayed resistance against colistin, while Citrobacter spp. and Klebsiella spp. displayed 90% and 60% resistance against this antimicrobial respectively. These findings outline the need for the development of new antimicrobials. About 75.5% (25/33) of the presumptive Enterobacter spp. were confirmed by MALDI-TOF with 79.2% (19/24), 66.7% (2/3), 66.7% (4/6) been confirmed vegetables, hospital effluents and river water samples respectively. Likewise, about 77.8% (21/27) were confirmed as Citrobacter spp. of which 92.3% (12/13), 66.7% (2/3) and 63.6% (7/11) were from vegetables, hospital effluents and river water samples respectively. These results show that the selected vegetables were highly contaminated with resistant bacteria and thus unsafe to consume uncooked vegetable. Also river water was higly contaminated with resistant microbes, which also shows that these rivers are not fit to be used 17 as drinking water sources and recreational activities. Colistin is an antimicrobial used as a last resort of antibiotics because it exhibits broad-spectrum activity. However from the findings of the work at present, this is no longer the case. The spectrum of this antimicrobial is now reduced by Enterobacteriaceae members. To the best of my knowledge; relatively few resources have been provided to understanding, preventing, and controlling increasing antimicrobial resistance on global, national and local levels.
- Full Text:
- Date Issued: 2018
- Authors: Mpaka, Lindelwa
- Date: 2018
- Subjects: Enterobacteriaceae
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/14549 , vital:40003
- Description: The worldwide problem of antimicrobial resistance has limited the spectrum of the current affordable and effective antimicrobials. Infections associated with resistant microorganisms impose a major threat to public health and economic stability. Globally, about 700 000 deaths every year can be accredited to antimicrobial resistance. The leading mechanism of resistance amid bacterial pathogens is the extended spectrum beta-lactamases production, which inhibits spectrum activity of several antimicrobial agents. The rise in antimicrobial resistance has compelled an urgent need of developing means of combatting resistance issue amid diseasecausing microbes. The main aim of this study is to evaluate the incidence and antibiogram fingerprints of Enterobacteriaceae recovered from hospital effluents, river water and vegetables in the Eastern Cape Province. A total of eighteen antibiotics from ten different antimicrobial classes were used to determine antibiogram profiles of the MALDI-TOF confirmed isolates. From the MALDI-TOF confirmed isolates, 60% of Enterobacter spp. and E. coli isolates displayed resistance against colistin, while Citrobacter spp. and Klebsiella spp. displayed 90% and 60% resistance against this antimicrobial respectively. These findings outline the need for the development of new antimicrobials. About 75.5% (25/33) of the presumptive Enterobacter spp. were confirmed by MALDI-TOF with 79.2% (19/24), 66.7% (2/3), 66.7% (4/6) been confirmed vegetables, hospital effluents and river water samples respectively. Likewise, about 77.8% (21/27) were confirmed as Citrobacter spp. of which 92.3% (12/13), 66.7% (2/3) and 63.6% (7/11) were from vegetables, hospital effluents and river water samples respectively. These results show that the selected vegetables were highly contaminated with resistant bacteria and thus unsafe to consume uncooked vegetable. Also river water was higly contaminated with resistant microbes, which also shows that these rivers are not fit to be used 17 as drinking water sources and recreational activities. Colistin is an antimicrobial used as a last resort of antibiotics because it exhibits broad-spectrum activity. However from the findings of the work at present, this is no longer the case. The spectrum of this antimicrobial is now reduced by Enterobacteriaceae members. To the best of my knowledge; relatively few resources have been provided to understanding, preventing, and controlling increasing antimicrobial resistance on global, national and local levels.
- Full Text:
- Date Issued: 2018
A structural study of the capsular antigen of Klebsiella serotype K43
- Authors: Aereboe, Michael
- Date: 1993
- Subjects: Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3740 , http://hdl.handle.net/10962/d1003218 , Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Description: This thesis presents a detailed chemical and spectroscopic determination of the capsular, polysaccharide K-antigen isolated from the Klebsiella bacterium, serotype K43 (culture #2482). The repeating unit of the capsular polysaccharide was found to be of the "3 + 2" repeating unit type. A uronic acid was found as part of a disaccharide side chain and the main chain of the polysaccharide was found to be composed of a neutral trisaccharide of mannose and galactose. The work forms part of an ongoing research interest in bacterial polysaccharides of this laboratory and now completes the structural elucidation of all the Klebsiella K-antigens, bar three antigens which were originally assigned to other laboratories. These data together with the respective serological characteristics of each serotype are available to the molecular biologist, and may result in the production of: vaccine(s) against Klebsiella infections, diagnostic products and novel carrier molecules enabling targeted drug delivery.
- Full Text:
- Date Issued: 1993
- Authors: Aereboe, Michael
- Date: 1993
- Subjects: Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3740 , http://hdl.handle.net/10962/d1003218 , Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Description: This thesis presents a detailed chemical and spectroscopic determination of the capsular, polysaccharide K-antigen isolated from the Klebsiella bacterium, serotype K43 (culture #2482). The repeating unit of the capsular polysaccharide was found to be of the "3 + 2" repeating unit type. A uronic acid was found as part of a disaccharide side chain and the main chain of the polysaccharide was found to be composed of a neutral trisaccharide of mannose and galactose. The work forms part of an ongoing research interest in bacterial polysaccharides of this laboratory and now completes the structural elucidation of all the Klebsiella K-antigens, bar three antigens which were originally assigned to other laboratories. These data together with the respective serological characteristics of each serotype are available to the molecular biologist, and may result in the production of: vaccine(s) against Klebsiella infections, diagnostic products and novel carrier molecules enabling targeted drug delivery.
- Full Text:
- Date Issued: 1993
Structural analysis of some Escherichia coli capsular antigens
- Authors: Hackland, Peter Linton
- Date: 1992
- Subjects: Antigens , Enterobacteriaceae , Escherichia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3758 , http://hdl.handle.net/10962/d1003236 , Antigens , Enterobacteriaceae , Escherichia
- Description: The work presented in this thesis forms part of a collaborative effort to determine the chemical structures of the surface antigens of bacteria which belong to the Enterobacteriaceae. These antigens are largely polysaccharides and occur as lipopolysaccharides and capsular polysaccharides which give rise to the somatic or 0 antigens and the capsular or K antigens, respectively. In recent years interest has mostly been focused on the extracellular polysaccharide antigens expressed by the genus Escherichia coli because of the effect they exert on normal immunological processes and their structural relatedness to the surface antigens of other more pathogenic bacteria. Therefore the molecular structures of the capsular polysaccharides (Kantigens)produced by E. coli 09:K35(AI04a) and 09:K38(A262a) have been determined by novel enzymic, chemical and spectroscopic procedures. These investigations show that the structures of these polysaccharides can be determined by a combination of chemical and spectroscopic procedures , or almost entirely by n.m.r. spectroscopy alone. The in vitro bacteriophage mediated depolymerisation of the native E. coli K35 polysaccharide demonstrates the value of this method for the isolation of oligosaccharides representing the repeating- unit and multiples thereof. Finally E. coli K37 and K38 capsular polysaccharides were used as model compounds for the evaluation of partial and selective reductive cleavage as methods of generating oligosaccharide for further structural analysis. The products of these reactions were analysed largely by a combination of mass spectrometric procedures.
- Full Text:
- Date Issued: 1992
- Authors: Hackland, Peter Linton
- Date: 1992
- Subjects: Antigens , Enterobacteriaceae , Escherichia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3758 , http://hdl.handle.net/10962/d1003236 , Antigens , Enterobacteriaceae , Escherichia
- Description: The work presented in this thesis forms part of a collaborative effort to determine the chemical structures of the surface antigens of bacteria which belong to the Enterobacteriaceae. These antigens are largely polysaccharides and occur as lipopolysaccharides and capsular polysaccharides which give rise to the somatic or 0 antigens and the capsular or K antigens, respectively. In recent years interest has mostly been focused on the extracellular polysaccharide antigens expressed by the genus Escherichia coli because of the effect they exert on normal immunological processes and their structural relatedness to the surface antigens of other more pathogenic bacteria. Therefore the molecular structures of the capsular polysaccharides (Kantigens)produced by E. coli 09:K35(AI04a) and 09:K38(A262a) have been determined by novel enzymic, chemical and spectroscopic procedures. These investigations show that the structures of these polysaccharides can be determined by a combination of chemical and spectroscopic procedures , or almost entirely by n.m.r. spectroscopy alone. The in vitro bacteriophage mediated depolymerisation of the native E. coli K35 polysaccharide demonstrates the value of this method for the isolation of oligosaccharides representing the repeating- unit and multiples thereof. Finally E. coli K37 and K38 capsular polysaccharides were used as model compounds for the evaluation of partial and selective reductive cleavage as methods of generating oligosaccharide for further structural analysis. The products of these reactions were analysed largely by a combination of mass spectrometric procedures.
- Full Text:
- Date Issued: 1992
A structural study of the capsular antigens of escherichia coli K36 and klebiella K68
- Authors: Stanley, Shawn Mark Ross
- Date: 1987 , 2013-03-11
- Subjects: Enterobacteriaceae , Klebsiella , Escherichia , Antigens
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3814 , http://hdl.handle.net/10962/d1004613 , Enterobacteriaceae , Klebsiella , Escherichia , Antigens
- Description: From Introduction: Bacterial cells all have a cytoplasmic membrane (see Figure 1) which regulates the movement of ions and molecules into and out of the bacterium. Enclosing this membrane is a cell wall of which there are two general types, which are differentiated by the Gram stain(02) as being either gram positive or gram negative (depending upon whether they hold the gram stain after washing with ethanol). The cell wall provides the cell with shape and rigidity and is composed, in the case of gram positive types, of peptidoglycan, and in the case of gram negative bacteria, of a peptidoglycan and an outer membrane (see Figure 2). The peptidoglycan layer, common to both cell wall types, consists of a backbone of alternating units of N-acetylglucosamine and N-acetylmuramic acid to which peptides are attached by amide links. This heteropolymer is a highly cross linked mosaic and this gives it strength and rigidity. In gram positive bacteria, this layer also contains two carbohydr ate antigens, a simple polysaccharide and a teichoic acid; these are usually the type specific or major group antigens of the bacterium. Many of the bacteria also produce exopolysaccharides (see Figure 3) either as discrete capsules (for example, the Enterobacteriaceae K antigens) or unattached slime layers (for example, the Enterobacteriaceae M antigens). The vast majority of these polysaccharides are heteroglycans(03) composed of contiguous oligosaccharide repeating units. Their monosaccharide components are largely neutral hexoses, 6-deoxy hexoses and also amino sugars. (03) Pentose units are rare. (03) The capsular polysaccharides usually have a high content of acidic constituents such as uronic acids, phosphate groups, or pyruvate ketals. (01) , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1987
- Authors: Stanley, Shawn Mark Ross
- Date: 1987 , 2013-03-11
- Subjects: Enterobacteriaceae , Klebsiella , Escherichia , Antigens
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3814 , http://hdl.handle.net/10962/d1004613 , Enterobacteriaceae , Klebsiella , Escherichia , Antigens
- Description: From Introduction: Bacterial cells all have a cytoplasmic membrane (see Figure 1) which regulates the movement of ions and molecules into and out of the bacterium. Enclosing this membrane is a cell wall of which there are two general types, which are differentiated by the Gram stain(02) as being either gram positive or gram negative (depending upon whether they hold the gram stain after washing with ethanol). The cell wall provides the cell with shape and rigidity and is composed, in the case of gram positive types, of peptidoglycan, and in the case of gram negative bacteria, of a peptidoglycan and an outer membrane (see Figure 2). The peptidoglycan layer, common to both cell wall types, consists of a backbone of alternating units of N-acetylglucosamine and N-acetylmuramic acid to which peptides are attached by amide links. This heteropolymer is a highly cross linked mosaic and this gives it strength and rigidity. In gram positive bacteria, this layer also contains two carbohydr ate antigens, a simple polysaccharide and a teichoic acid; these are usually the type specific or major group antigens of the bacterium. Many of the bacteria also produce exopolysaccharides (see Figure 3) either as discrete capsules (for example, the Enterobacteriaceae K antigens) or unattached slime layers (for example, the Enterobacteriaceae M antigens). The vast majority of these polysaccharides are heteroglycans(03) composed of contiguous oligosaccharide repeating units. Their monosaccharide components are largely neutral hexoses, 6-deoxy hexoses and also amino sugars. (03) Pentose units are rare. (03) The capsular polysaccharides usually have a high content of acidic constituents such as uronic acids, phosphate groups, or pyruvate ketals. (01) , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1987
Studies on the ecology and molecular biology of transferable drug resistance factors in coliform bacteria
- Authors: Marcos, David
- Date: 1973
- Subjects: Enterobacteriaceae , Molecular biology , Microbial ecology , Bacteria -- Ecology , Ecology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4249 , http://hdl.handle.net/10962/d1007494 , Enterobacteriaceae , Molecular biology , Microbial ecology , Bacteria -- Ecology , Ecology
- Description: From Introduction: It was as early as 1904 that Paul Ehrlich propounded the idea of a “magic bullet”. This “magic bullet”, or chemotherapeutic agent, as he also called it, had to meet certain requirements: (a) a high activity against pathogenic micro-organisms; (b) easy absorption by the body; (c) activity in the presence of body fluids and tissue; (d) a low degree of toxicity; (e) must not allow the development of resistant micro-organisms. The discovery of the sulphonamide, Prentosil, by Domagk in 1935 was one of the initial steps in the search for this “magic bullet”. This, together with the production and purification of the antibiotics penicillin, by Fleming, Florey and Chain in 1942 and streptomycin, by Waksman in 1943, heralded a new era in the fight against bacterial infections. The majority of modern antibacterial agents have to a large extent met the requirements of Ehrlich’s ‘magic bullet”. They have however failed to prevent the development of resistant bacterial strains. This has been particularly noticeable in the past twenty years since the sudden emergence of multiple-resistant bacteria, many of which can transfer to several drugs in one step by a process of conjugation. This phenomenon which has serious medical implications has prompted numerous studies on the origin, epidemiology, biochemistry and genetics of transferable drug resistance.
- Full Text:
- Date Issued: 1973
- Authors: Marcos, David
- Date: 1973
- Subjects: Enterobacteriaceae , Molecular biology , Microbial ecology , Bacteria -- Ecology , Ecology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4249 , http://hdl.handle.net/10962/d1007494 , Enterobacteriaceae , Molecular biology , Microbial ecology , Bacteria -- Ecology , Ecology
- Description: From Introduction: It was as early as 1904 that Paul Ehrlich propounded the idea of a “magic bullet”. This “magic bullet”, or chemotherapeutic agent, as he also called it, had to meet certain requirements: (a) a high activity against pathogenic micro-organisms; (b) easy absorption by the body; (c) activity in the presence of body fluids and tissue; (d) a low degree of toxicity; (e) must not allow the development of resistant micro-organisms. The discovery of the sulphonamide, Prentosil, by Domagk in 1935 was one of the initial steps in the search for this “magic bullet”. This, together with the production and purification of the antibiotics penicillin, by Fleming, Florey and Chain in 1942 and streptomycin, by Waksman in 1943, heralded a new era in the fight against bacterial infections. The majority of modern antibacterial agents have to a large extent met the requirements of Ehrlich’s ‘magic bullet”. They have however failed to prevent the development of resistant bacterial strains. This has been particularly noticeable in the past twenty years since the sudden emergence of multiple-resistant bacteria, many of which can transfer to several drugs in one step by a process of conjugation. This phenomenon which has serious medical implications has prompted numerous studies on the origin, epidemiology, biochemistry and genetics of transferable drug resistance.
- Full Text:
- Date Issued: 1973
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