plasmid

摘要： We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou,China.A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes.Characterization of bla_(NDM-5) positive isolates and plasmids was determined by antimicrobial susceptibility testing,conjugation experiments,Illumina HiSeq,and Nanopore sequencing.One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as bla_(NDM-5) carriers(3.57%,7/196).The bla_(NDM-5) genes were located on the lncX3(n=5),lncHI2(n=1),or lncHI2-lncF(n=1)plasmids.All bla_(NDM-5)-bearing plasmids were transferred by conjugation at frequencies of~10^(-4)-10^(-6).Based on sequence analysis,the lncHI2 plasmid pHNBYF33-1 was similar to other bla_(NDM-5)-carrying lncHI2 plasmids deposited in GenBank from Guangdong ducks.In all lncHI2 plasmids,bla_(NDM-5)was embedded in a novel transposon,Tn7057(IS3000-△ISAba125-IS5-△ISAba125-bla_(NDM-5)-bleMBL-trpF-tat-△dct-IS26-△umuD-△ISKox3-IS3000),which was identical to the genetic structure surrounding bla_(NDM-5)found in some IncX3 plasmids.The lncHI2-lncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the bla_(NDM-5)-carrying lncHI2 plasmid and a heavy-metal-resistant IncF plasmid through△Tn1721 To the best of our knowledge,this is the first report on the characterization of bla_(NDM-5)-bearing plasmids in fish in China.The lncHI2 plasmid pHNBYF33-1 may be transmitted from ducks,considering the common duck-fish freshwater aquaculture system in Guangdong.Tn7051 is likely responsible for the transfer of bla_(NDM-5) from lncX3 to lncHI2 plasmids in Enterobacteriaceae,resulting in the expansion of transmission vectors of bla_(NDM-5).

摘要： 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.

摘要： The increasing incidence of multidrug-resistant Klebsiella pneumoniae strains has become a serious global healthcare problem. Additionally, the carriage of both extended-spectrum ß-lactamase and carbapenemase genes on plasmid and genomic DNA in K. pneumoniae clinical isolates has not been documented in Kenya. This study aimed to assess the presence of extended spectrum β-lactamase (ESBL) and carbapenemase genes on genomic and plasmid DNA in K. pneumoniae, and classify these super-bug clinical isolates based on their phylogenetic patterns. The identification of Klebsiella-like clinical isolates (n = 20) collected from Kenyatta National Hospital in Nairobi was performed using API 20E Kit. Screening and confirmation for ESBL and carbapenemase phenotypes were conducted using Kirby-Bauer disk diffusion susceptibility test protocol. Conventional PCR technique was used to characterize ESBL and carbapenemase resistant genes on both genomic and plasmid DNA. Subsequently, 16S rRNA gene amplification and sequencing were performed. The 16S rRNA gene contiguous sequences of the bacterial isolates were analyzed using the ChromasPro. The gene sequence was compared with the sequences in GenBank database, using the BLAST program of NCBI to obtain the nearest phylogenetic neighbours from the databases. Then, the sequences of MDR K. pneumoniae and its relatives were aligned using ClustalW. The evolutionary history was inferred by using the maximum likelihood algorithm in MEGA MX. The phenotypic data of antibiotic susceptibility testing revealed that 2/20 (10%) clinical isolates were resistant both to imipenem and meropenem and producers of carbapenemase. These isolates were carbapenemase producers but not extended β-lactamases. However, 3/20 (15%) isolates that co-harboured blaNDM-1, blaIMP, blaTEM, and bla-OXA were identified during genotypic analysis. The positive control used separately yielded the expected band sizes for blaIMP (275 bp), blaOXA-48 (438 bp), and BlaKPC (798). The phylogenetic analysis showed the dual ESBL and carbapenemase producing Klebsiella pneumoniae could be classified as K. pneumoniae strain DSM 30104 and K. pneumonia subsp. pneumoniae strain GMH1080. This study confirmed the co-existence of ESBL and carbapenemase genes in Klebsiella pneumoniae on both bacterial genomic and Plasmid DNA, and demonstrated that the isolates are evolutionarily distinct. These findings raise a concern about the genotypic diversity of antibiotic resistance genes in bacterial isolates and their location. We, therefore, recommend an alternative management approach to combat these MDR bacterial isolates as well as frequent molecular surveillance programs to support antimicrobial stewardship.

摘要： A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total of 44 Edwardsiella isolates were recovered from 80 fish samples collected from the 10 fish farms using selective random stratification. It was observed that Edwardsiella tarda isolates were 100% resistant to Amoxicillin, Chloranphenicol, Levofloxacin, Streptomycin and 90% resistant to Nalidixic Acid respectively. All the isolates were 100% susceptible to Spectinomycin and Ciprofloxacin, while Ofloxacin, Gentamycin, and Pefloxacin vary in their level of susceptibility with 90%, 80% and 70% sensitivity respectively. Conversely, 8 out of 10 fish farm locations studied were observed to have antibiotic-resistant strains, and 5 out of 8 drug-resistant strains were found to carry plasmid and the sizes of the plasmid ranges between 20.027 kb to 23.130 kb. The plasmid after treatment with mitomycin C and ethidium bromide were lost during the process of plasmid curing confirming that the multiple drug resistant exhibited by the isolates was plasmid mediated. There are fewer studies on antibiotic resistance in Edwardsiella tarda from aquaculture enterprises and this study provides further support to the view that there is a potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Although, in Nigeria there is no antibiotic product registered for aquaculture usage, yet fish farmers use them off-label for bacterial diseases prevention.

摘要： Introduction: Salmonella enterica Serovars remains one of the leading pathogens that cause diarrhoea and bloodstream infections in developing countries. The emergence of multidrug resistant (MDR) Salmonella has become a serious problem globally. This study investigated the antibiotic resistance and plasmid profiles of Salmonella isolates from different sources. Methods: Seventy-three samples comprised of clinical (30), hand swab (15), food (10) and water (18) were analyzed bacteriologically. Salmonella isolates were identified and characterized by standard procedures. Isolates were subjected to antimicrobial susceptibility testing and were further screened for plasmid DNA by standard methods. Results: A total of 27 Salmonella isolates made up of 5 (18.5%) S. typhi, 6 (22.2%) S. enteritidis, 9 (33.3) S. typhimurium, 5 (18.5%) S. cholerasuis, and 1 (3.7%) each of S.arizonae and S. vichow were obtained in this study. All the isolates developed resistance to three or more antibiotics evaluated. Four distinct resistance profiles: TetAmpCol, TetAmpColCot, TetAmpColCip and TetAmpColCotCip were recorded with 63% of the isolates exhibiting resistance profile TetAmpColCot. Specifically 23 of 27 (85.2%) of the isolates harboured plasmid DNA comprised of 12 distinct plasmid profiles of different sizes ranging from 3.2 kb to 30.2kb. Salmonella isolates of the same species from different sources differed in plasmid profile. Plasmid profile was found to show good discriminatory capability compared to antibiotics resistance profile. Conclusion: This study revealed that both resistance antibiogram and plasmid profile are still viable epidemiological tools for tracing the source of Salmonella isolates. A need for prudent use of antibiotics is suggested.

摘要： Salmonella enterica serovar Rissen has been recognised as a common serovar in humans and pigs around the world. This study investigated S. Rissen prevalence in pigs slaughtered in Northern Ireland additionally looking at antibiotic susceptibility profiles, genetic profiles and plasmid profiles to provide information on an emerging non-typhoid Salmonella serotype with the potential to cause disease in humans. S. Rissen were isolated on five separate sampling occasions from both the boning hall and slaughter line of a randomly selected single pig abattoir in Northern Ireland (NI). Following antibiotic susceptibility testing against 16 antibiotics, all S. Rissen isolates were identified as susceptible to 15 antibiotics but resistant to tetracycline (R-type: Te). Of the 29 S. Rissen, 27 were isolated from pigs originating in NI and two S. Rissen were isolated from pigs originating in the Republic of Ireland (RoI). The combined results of the PFGE and plasmid profiling analyses were capable of subdividing the S. Rissen isolates into three distinct groups. The data suggests that S. Rissen is an emerging serovar in Northern Ireland and continued surveillance of this serovar is warranted as it has the potential to cause disease in humans.

摘要： The main component of the Center for Genetic Engineering and Biotechnology(CIGB)candidate vaccine against Hepatitis C virus(HCV)is the pIDKE2 plasmid.The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharmaceuticalgrade plasmid DNA(pDNA)with 95%purity.The advantages of this procedure include high plasmid purity and the elimination of undesirable additives,such as toxic organic extractants and animal-derived enzymes.However,yields and consequently the productivity of the process are low.Previous work demonstrated that the most critical step of the process is the reverse phase chromatography,where conventional porous particle resins are used.Therefore,to increase the process productivity,alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step.Here,a comparison between the behaviors of CIM~ C4-HLD and Sartobind phenyl matrices was performed.To obtain higher productivities and purities,the dynamic binding capacities and selectivities were evaluated.The results showed that both matrices had a similar capacity for pIDKE2 plasmid,but the separation of pDNA isoforms using CIM~ technology was much better than that with Sartobind.Additionally,the optimal conditions for loading plasmid DNA on a CIMC4-HLD 800-mL monolithic column in a real production process were determined.These optimizations will allow production levels to satisfy the high plasmid consumption demanded by clinical trials.

摘要： Background: Extended spectrum beta-lactamases (ESBLs) are enzymes that compromise the efficacy of all beta-lactams and are spread by plasmids. They are of public health importance the world over;however, in Nigeria in general and Uyo in particular, tests for their detection are not routinely done in hospital laboratories despite increase in treatment failures observed for common clinical conditions like urinary tract infection. Objective: To isolate ESBLs producing uropathogens and the plasmid underlying their resistance to antibiotics. Materials and Methods: Three hundred urine specimens (n = 300) were collected from pregnant women attending antenatal clinics at St. Lukes Hospital, Anua, cultured and incubated according to accepted standard. Identification of isolates was done using Microbact 24E (Oxoid, UK) system. The predominant bacterial pathogens were Escherichia coli (42%) followed by Klebsiella pneumonia (21%), Klebsiella oxytoca (12%), Citrobacter spp. (5%), Proteus mirabilis (7%), Enterobacter spp. (12%) and Acinetobacter baumanii (1%). The isolated bacteria were tested for their antibiotic susceptibility using Clinical Laboratory Standard Institute (CLSI) recommended disc diffusion method. A Double Disk Synergy Test (DDST) and Phenotypic Disk Confirmatory Test (PDCT) were performed to determine ESBL production. Chromagar ESBL was also used to test for the presence of ESBL producing isolates. The plasmid content of ESBL producing isolates and their participation in drug resistance were investigated. Results: Of the 80 bacterial isolates causing urinary tract infection in these women, the ESBL producers were found to be 16 (20%). Out of these 16 ESBL producing urogenital isolates Klebsiella pneumonia (8, 50%) was the most prevalent. Others include Escherichia coli (38%), Klebsiella oxytoca (6%) and Enterobacter cloacae (6). Plasmid content of ESBL producing isolates was found to be 87.5%. Conclusion: The Extended Spectrum Beta-lactamase producing uropathogens mainly of plasmid origin are increasingly responsible for the cause of community acquired urinary tract infections in pregnant women in Uyo.

摘要： 背景：在基因治疗中选择合适、低毒、对人体和环境无害的载体，使基因高效地转移至靶向部位并有效表达相关产物尤为关键。目的：制备超顺磁性Fe3O4/SiO2-聚乙酰亚胺复合微球。方法：通过乳化溶剂挥发法制备Fe3O4纳米粒子聚集体，再利用stober法合成超顺磁性Fe3O4/SiO2核壳型微球，进一步在该微球表面修饰聚乙酰亚胺，得到超顺磁性Fe3O4/SiO2-聚乙酰亚胺复合微球，并对其进行透射电镜、Zeta电位和磁性等结构性能表征。将Fe3O4/SiO2-聚乙酰亚胺复合微球与Plasmid DNA按照不同的质量比（29∶1，39∶1，49∶1，59∶1，68∶1，78∶1，88∶1）混合，通过凝胶电泳测定该复合微球与绿色荧光蛋白基因的结合能力。将Plasmid DNA分别与Fe3O4/SiO2-聚乙酰亚胺、聚乙酰亚胺混合，通过共聚焦荧光显微镜观测其在HeLa细胞中转染绿色荧光蛋白基因的情况。结果与结论：成功合成了Fe3O4/SiO2-聚乙酰亚胺复合微球，分散性良好，粒径分布均匀，约为100 nm，表面电荷为21.07 mV，饱和磁化强度为28.05 emu/g，为超顺磁性。随着复合微球与Plasmid DNA质量比的不断增加，越来越多的Plasmid DNA质粒被吸附在Fe3O4/SiO2-聚乙酰亚胺复合微球上，此时Plasmid DNA质粒过量，当质量比达到59∶1时，所有的pDNA质粒都被吸附在复合微球上；质量比大于59∶1时，复合微球过量，因此质量比为59∶1时二者均无过量，结果较好，用于 HeLa 细胞转染。与聚乙酰亚胺相比， Fe3O4/SiO2-聚乙酰亚胺复合微球可显著提高Plasmid DNA的转染效率。

摘要： 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.