Multi-drug resistance (MDR) in Enterobacteriaceae poses critical public health threat in Nigeria and the global world. This resistant mechanism might be plasmid mediated or chromosomal. Escherichia coli are Gram negat...Multi-drug resistance (MDR) in Enterobacteriaceae poses critical public health threat in Nigeria and the global world. This resistant mechanism might be plasmid mediated or chromosomal. Escherichia coli are Gram negative pathogen with a global distribution rate. The study was carried out to determine MDR and plasmid profiling of E. coli isolates from urine, feaces and poultry litter. The samples were cultured on eosine methylene blue agar and incubated for 24 hours at 37°C. Results obtained showed a percentage prevalence of 30% for the urine samples which were the most prevalent, while the prevalence of E. coli from the feacal and poultry litter was 8% and 28% respectively. Identified E. coli were screened for antibiotic susceptibility by Kirby Bauer diffusion method. The results on susceptibility of E. coli to tested antibiotics before plasmid curing showed 100% resistance to cefuroxime and augumentin, while 75% resistance was observed in gentamicine, ciprofloxacin and ofloxacine. Cefixime and cefdazidime resistance were 62.5% on E. coli and the least resistance was observed in nitrofurantion (25%). The poultry litter and urine isolates recorded lower resistance level to antibiotics, compared to the feacal isolates. After plasmid curing the percentage of resistance reduced. The only antibiotics that responded positively was nitrofurantion, with high sensitivity of 87% for feacal isolate, 100% for urine isolates, and 78% for poultry litter isolates after plasmid curing. Twenty (20) of the thirty seven (37) isolates were still resistant to more than two antibiotics after the plasmid curing. Of the twenty isolates, 18 (90%) were found to harbor single plasmid, while 2 (10%) did not possess plasmid. This study concludes that nitrofurantion was the most effective antibiotics on Escherichia coli and plasmids were responsible partly for resistance.展开更多
Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this meth...Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.展开更多
Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been id...Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been identified as reservoirs for broad-host-range plasmids carrying resistance genes. The threat of broad-host-range plasmids released into the environment from wastewater treatment plants has identified the need for disinfection protocols to target broad-host- range plasmid destruction. Here we evaluate the efficacy of dissolved ozone at 2 and 8 mg·L–1 as a primary means for the destruction of broad-host-range plasmid and chromosomal DNA in simulated effluent. Pilot-scale tests using an experimental unit were carried out in municipal wastewater treatment plant effluent and compared with ultraviolet (UV)-irradiation and chlorination methodologies. Genes specific to Escherichia coli (uidA) and IncP broad-host-range plasmids (trfA) were monitored using real-time quantitative polymerase chain reaction (qPCR), and total DNA was monitored using absorbance spectroscopy. In wastewater treatment plant experiments, E. coli qPCR results were compared to a recognized culture-based method (Colilert?) for E. coli. In laboratory experiments, dissolved ozone at 8 mg·L–1 significantly destroyed 93% total, 98% E. coli, and 99% of broad-host-range plasmid DNA. Ozonation, UV-irradiation, and chlorination significantly reduced DNA concentrations and culturable E. coli in wastewater treat- ment plant effluent. Chlorination and UV disinfection resulted in 3-log decreases in culture-based E. coli concentrations in wastewater treatment plant effluent while changes were not significant when measured with qPCR. Only ozonation significantly decreased the IncP broad-host-range plasmid trfA gene, although concentrations of 2.2 × 105 copies trfA·L–1 remained in effluent. Disinfection processes utilizing high dissolved ozone concentrations for the destruction of emerging contaminants such as broad-host-range plasmid and total DNA may have utility as methods to ensure downstream environmental health and safe water reuse become more important.展开更多
Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli is a global cause of life threatening infections. We determined the presence of ESBL and carbapenemase production in clinical isolates of E. coli and ...Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli is a global cause of life threatening infections. We determined the presence of ESBL and carbapenemase production in clinical isolates of E. coli and their antibiotic susceptibility. Clinical isolates of community and hospital acquired E. coli from 220 patients seen at a tertiary hospital were evaluated. Antibiotic susceptibility testing was by the modified Kirby-Bauer protocol while ESBL production was determined by the Double Disk Synergy Test (DDST). Carbapenem resistance was confirmed by the Modified Hodge Test. Of the 220 isolates, 122 (55.5%) were from females;41 (18.6%) were ESBL positive. About 90% of the ESBL producing isolates were resistant to nine of the 15 antimicrobial agents tested. However, only one (2.4%) of the 41 ESBL producing isolates exhibited carbapenem resistance. The ESBL negative isolates were susceptible to Meropenem (100%), Cefepime (97.8%), Ceftriaxone (96.6%) and Cefotaxime (96.6%). All the ESBL producing isolates harbored detectable plasmids with sizes ranging from 2322 to 23,130 base pairs. Our findings show that although multidrug resistant ESBL producing E. coli are prevalent in both the hospital and the community in this environment, carbapenem resistance is still low. We recommend that institutions develop guidelines for the early phenotypic detection of ESBLs and carbapenem resistance.展开更多
Background: Extended spectrum β-lactamases (ESBL) producing E. coli co-producing other β-lactamases and exhibiting co-resistance to different antibiotic classes continue to emerge as a threat to clinical field. This...Background: Extended spectrum β-lactamases (ESBL) producing E. coli co-producing other β-lactamases and exhibiting co-resistance to different antibiotic classes continue to emerge as a threat to clinical field. This study aimed to analyze the co-production of New Delhi metallo-β-lactamase-1 (blaNDM-1) in ESBL producing plasmid-bearing clinical isolates collected from two tertiary care centres in Kerala, South India, and to understand their genetic relatedness. Methods: Antibiotic resistance phenotypes of 44 clinical isolates were determined by disc-diffusion method. Plasmid-bearing isolates, detected by the alkaline-lysis method, which also tested positive for ESBL production, were screened for the presence of blaNDM-1 by polymerase chain reaction. Plasmid, random amplified polymorphic DNA profiles and blaNDM-1 sequence-based phylogenetic tree were analyzed to understand the genotypic similarities among the isolates. Results: Beta-lactam antibiotics, quinolones, cephalosporins, used in this study, and AZM were found to be ineffective against the isolates as significantly high number of isolates were resistant to these antibiotics (P < 0.01). Plasmid bearing isolates constituted 57% (n = 25), all of which were found to be ESBL producers. blaNDM-1 amplicons were noticed in four (16%) isolates and these DNA sequences showed homology between them and with similar sequences reported from other countries like Japan and Korea. Plasmid and RAPD profiles demonstrated that most of the isolates, including those harbouring blaNDM-1 shared genetic similarities as well as an apparent geographical distinctiveness. Conclusion: The predominance of ESBL production and the occurrence of blaNDM-1 in plasmid-bearing isolates observed in our study corroborate the worldwide drug-resistance scenario. This study thus warrants the need for constant surveillance in the face of sparse information available in Kerala State on the emerging drug resistance in clinical bacteria.展开更多
Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of tran...Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of transformed Escherichia coli (E. coli) in liquid medium, brief centrifugation for precipitation of bacterial pellets, and subsequent lysis of the pellets. This process is time-consuming and laborious, especially when the sample number is high. Here, we describe a more convenient method for MiniPrep analysis that utilizes solid medium-based cultivation of bacteria.展开更多
文摘Multi-drug resistance (MDR) in Enterobacteriaceae poses critical public health threat in Nigeria and the global world. This resistant mechanism might be plasmid mediated or chromosomal. Escherichia coli are Gram negative pathogen with a global distribution rate. The study was carried out to determine MDR and plasmid profiling of E. coli isolates from urine, feaces and poultry litter. The samples were cultured on eosine methylene blue agar and incubated for 24 hours at 37°C. Results obtained showed a percentage prevalence of 30% for the urine samples which were the most prevalent, while the prevalence of E. coli from the feacal and poultry litter was 8% and 28% respectively. Identified E. coli were screened for antibiotic susceptibility by Kirby Bauer diffusion method. The results on susceptibility of E. coli to tested antibiotics before plasmid curing showed 100% resistance to cefuroxime and augumentin, while 75% resistance was observed in gentamicine, ciprofloxacin and ofloxacine. Cefixime and cefdazidime resistance were 62.5% on E. coli and the least resistance was observed in nitrofurantion (25%). The poultry litter and urine isolates recorded lower resistance level to antibiotics, compared to the feacal isolates. After plasmid curing the percentage of resistance reduced. The only antibiotics that responded positively was nitrofurantion, with high sensitivity of 87% for feacal isolate, 100% for urine isolates, and 78% for poultry litter isolates after plasmid curing. Twenty (20) of the thirty seven (37) isolates were still resistant to more than two antibiotics after the plasmid curing. Of the twenty isolates, 18 (90%) were found to harbor single plasmid, while 2 (10%) did not possess plasmid. This study concludes that nitrofurantion was the most effective antibiotics on Escherichia coli and plasmids were responsible partly for resistance.
文摘Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.
文摘Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been identified as reservoirs for broad-host-range plasmids carrying resistance genes. The threat of broad-host-range plasmids released into the environment from wastewater treatment plants has identified the need for disinfection protocols to target broad-host- range plasmid destruction. Here we evaluate the efficacy of dissolved ozone at 2 and 8 mg·L–1 as a primary means for the destruction of broad-host-range plasmid and chromosomal DNA in simulated effluent. Pilot-scale tests using an experimental unit were carried out in municipal wastewater treatment plant effluent and compared with ultraviolet (UV)-irradiation and chlorination methodologies. Genes specific to Escherichia coli (uidA) and IncP broad-host-range plasmids (trfA) were monitored using real-time quantitative polymerase chain reaction (qPCR), and total DNA was monitored using absorbance spectroscopy. In wastewater treatment plant experiments, E. coli qPCR results were compared to a recognized culture-based method (Colilert?) for E. coli. In laboratory experiments, dissolved ozone at 8 mg·L–1 significantly destroyed 93% total, 98% E. coli, and 99% of broad-host-range plasmid DNA. Ozonation, UV-irradiation, and chlorination significantly reduced DNA concentrations and culturable E. coli in wastewater treat- ment plant effluent. Chlorination and UV disinfection resulted in 3-log decreases in culture-based E. coli concentrations in wastewater treatment plant effluent while changes were not significant when measured with qPCR. Only ozonation significantly decreased the IncP broad-host-range plasmid trfA gene, although concentrations of 2.2 × 105 copies trfA·L–1 remained in effluent. Disinfection processes utilizing high dissolved ozone concentrations for the destruction of emerging contaminants such as broad-host-range plasmid and total DNA may have utility as methods to ensure downstream environmental health and safe water reuse become more important.
文摘Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli is a global cause of life threatening infections. We determined the presence of ESBL and carbapenemase production in clinical isolates of E. coli and their antibiotic susceptibility. Clinical isolates of community and hospital acquired E. coli from 220 patients seen at a tertiary hospital were evaluated. Antibiotic susceptibility testing was by the modified Kirby-Bauer protocol while ESBL production was determined by the Double Disk Synergy Test (DDST). Carbapenem resistance was confirmed by the Modified Hodge Test. Of the 220 isolates, 122 (55.5%) were from females;41 (18.6%) were ESBL positive. About 90% of the ESBL producing isolates were resistant to nine of the 15 antimicrobial agents tested. However, only one (2.4%) of the 41 ESBL producing isolates exhibited carbapenem resistance. The ESBL negative isolates were susceptible to Meropenem (100%), Cefepime (97.8%), Ceftriaxone (96.6%) and Cefotaxime (96.6%). All the ESBL producing isolates harbored detectable plasmids with sizes ranging from 2322 to 23,130 base pairs. Our findings show that although multidrug resistant ESBL producing E. coli are prevalent in both the hospital and the community in this environment, carbapenem resistance is still low. We recommend that institutions develop guidelines for the early phenotypic detection of ESBLs and carbapenem resistance.
文摘Background: Extended spectrum β-lactamases (ESBL) producing E. coli co-producing other β-lactamases and exhibiting co-resistance to different antibiotic classes continue to emerge as a threat to clinical field. This study aimed to analyze the co-production of New Delhi metallo-β-lactamase-1 (blaNDM-1) in ESBL producing plasmid-bearing clinical isolates collected from two tertiary care centres in Kerala, South India, and to understand their genetic relatedness. Methods: Antibiotic resistance phenotypes of 44 clinical isolates were determined by disc-diffusion method. Plasmid-bearing isolates, detected by the alkaline-lysis method, which also tested positive for ESBL production, were screened for the presence of blaNDM-1 by polymerase chain reaction. Plasmid, random amplified polymorphic DNA profiles and blaNDM-1 sequence-based phylogenetic tree were analyzed to understand the genotypic similarities among the isolates. Results: Beta-lactam antibiotics, quinolones, cephalosporins, used in this study, and AZM were found to be ineffective against the isolates as significantly high number of isolates were resistant to these antibiotics (P < 0.01). Plasmid bearing isolates constituted 57% (n = 25), all of which were found to be ESBL producers. blaNDM-1 amplicons were noticed in four (16%) isolates and these DNA sequences showed homology between them and with similar sequences reported from other countries like Japan and Korea. Plasmid and RAPD profiles demonstrated that most of the isolates, including those harbouring blaNDM-1 shared genetic similarities as well as an apparent geographical distinctiveness. Conclusion: The predominance of ESBL production and the occurrence of blaNDM-1 in plasmid-bearing isolates observed in our study corroborate the worldwide drug-resistance scenario. This study thus warrants the need for constant surveillance in the face of sparse information available in Kerala State on the emerging drug resistance in clinical bacteria.
文摘Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of transformed Escherichia coli (E. coli) in liquid medium, brief centrifugation for precipitation of bacterial pellets, and subsequent lysis of the pellets. This process is time-consuming and laborious, especially when the sample number is high. Here, we describe a more convenient method for MiniPrep analysis that utilizes solid medium-based cultivation of bacteria.
