Plasmid Screening Research Articles

Incidence of Multidrug Resistance R-plasmids among Escherichia coli Causing Urinary Tract Infections: A Case Study from Nigeria

Aims: Study on the antimicrobial resistance pattern and plasmid screening of some clinical strains of Escherichia coli isolated from urine of some urinary tract infection out patients. Study design: Cross-sectional study. Place and Duration of Study: General hospital Lagos Island, Lagos, Nigeria, between March 2009 and August 2009. Methodology: Fifty resistant strains of E. coli were isolated by standard procedures from 96 clinical specimens. Antimicrobial susceptibility testing and plasmid screening were done on the strains. Followed by plasmid isolation and gene transfer experiment. plasmid curing of the resistant plasmid was also carried out. Results: Out of 96 samples screened only 50 (52.1%) yielded clinical isolates of E. coli. Among the various classes of antibiotic tested, high resistance was found with amoxycillin (100%), followed by tetracycline (98%), and augmentin (98%), while nitrofurantoin, ceftriaxone and ofloxacin being the most potent with (90%), (58%), (58%), sensitivity respectively. All the strains that were resistant to any antimicrobial agent were also resistant to amoxycillin. 34.4% of the isolates with multiple antimicrobial Case Study British Journal of Applied Science & Technology, 1(4): 204-210, 2011 205 resistant haboured single plasmids ranging in sizes from 4.5kb to 4.8kb. Transformation experiment revealed that six of these resistant strains carried a common R-plasmid of size 4.8kb. Plasmid determined resistance to tetracycline was identified. Conclusion: This study has highlighted the emergence of multidrug resistance Rplasmids among Escherichia coli causing urinary tract infections in Nigeria. There is a high level of resistance to many antimicrobials that are frequently used in Nigeria. The uncontrolled use of antibiotics has contributed largely to this situation. Thus the government should make considerable effort to establish an antibiotic policy for the country.

Screening of degradative plasmids from Arthrobacter sp. HW08 and their ability to degrade swainsonine

Arthrobacter sp. HW08 is capable of swainsonine (SW) degradation with high efficiency. The aim of this study was to screen the degradative plasmids from Arthrobacter sp. HW08 and investigate their ability of degrading SW. A genomic library of Arthrobacter sp. HW08 was successfully constructed by BamHI restriction digestion of genomic DNA and pUC19 vector, ligation and transformation into DH5. Five plasmids of different sizes were screened from 100 genomic library strains, and were named as pUCSW-1, pUCSW-2, pUCSW-3, pUCSW-4 and pUCSW-5, and their insertion elements had sizes of 6 684, 5 093, 136, 1 452 and 2 082 bp, respectively. They contained 7 major open reading frames. SW (400 mg l -1 ), as only carbon source, cultivated with the mixture of the five plasmid-transformants was degraded within 6 h. The degrading capability was equivalent to that of strain HW08. SW degradative ability of minimum combinations was pUCSW-2, 3, 5 > pUCSW-1, 2, 4 > pUCSW-1, 2, 5 > pUCSW-1, 3, 5, which were all slower than that of strain HW08. Any two or single transformant had no degradative ability. The results of this study support the search of the key genes of SW degradation and the investigating mechanism of SW biodegradation or exploration of its metabolic pathway, and even construction of transgene engineering bacteria to protect animals from lesion when locoweed is consumed. Key words : Swainsonine, biodegradation, degradative plasmids, Arthrobacter sp. HW08.

Copper-resistant halophilic bacterium isolated from the polluted Maruit Lake, Egypt

To isolate and characterize copper-resistant halophilic bacteria from the polluted Maruit Lake, Egypt and identify the role of plasmids in toxic metal resistance. We isolated strain MA2, showing high copper resistance up to the 1.5 mmol l(-1) concentration; it was also resistant to other metals such as nickel, cobalt and zinc and a group of antibiotics. Partial 16S rRNA analysis revealed that strain MA2 belonged to the genus Halomonas. Copper uptake, measured by atomic absorption spectrophotometery, was higher in the absence of NaCl than in the presence of 0.5-1.0 mol l(-1) NaCl during 5-15 min of incubation. Cell fractionation and electron microscopic observation clarified that most of the copper accumulated in the outer membrane and periplasmic fractions of the cells. Plasmid screening yielded two plasmids: pMA21 (11 kb) and pMA22 (5 kb). Plasmid curing resulted in a strain that lost both the plasmids and was sensitive to cobalt and chromate but not copper, nickel and zinc. This cured strain also showed weak growth in the presence of 0.5-1.0 mol l(-1) NaCl. Partial sequencing of both plasmids led to the identification of different toxic metals transporters but copper transporters were not identified. The highest cell viability was found in the presence of 1.0 mol l(-1) NaCl at different copper concentrations, and copper uptake was optimal in the absence of NaCl. Plasmid pMA21 encoded chromate, cobalt, zinc and cadmium transporters, whereas pMA22 encoded specific zinc and RND (resistance, nodulation, cell division) efflux transporters as well as different kinds of metabolic enzymes. Copper resistance was mainly incorporated in the chromosome. Strain MA2 is a fast and efficient tool for copper bioremediation and the isolated plasmids show significant characteristics of both toxic metal and antibiotic resistance.

Plasmids captured in C. metallidurans CH34: defining the PromA family of broad-host-range plasmids

The self-transmissible, broad-host-range (BHR) plasmid pMOL98 was previously isolated from polluted soil using a triparental plasmid capture approach and shown to possess a replicon similar to that of the BHR plasmids pSB102 and pIPO2. Here, complete sequence analysis and comparative genomics reveal that the 55.5 kb nucleotide sequence of pMOL98 shows extensive sequence similarity and synteny with the BHR plasmid family that now includes pIPO2, pSB102, pTER331, and pMRAD02. They share a plasmid backbone comprising replication, partitioning and conjugative transfer functions. Comparison of the variable accessory regions of these plasmids shows that the majority of natural transposons, as well as the mini-transposon used to mark the plasmids, are inserted in the parA locus. The transposon unique to pMOL98 appears to have inserted from the chromosome of the recipient strain used in the plasmid capture procedure. This demonstrates the necessity for careful screening of plasmids and host chromosomes to avoid mis-interpretation of plasmid genome content. The presence of very similar BHR plasmids with different accessory genes in geographically distinct locations suggests an important role in horizontal gene exchange and bacterial adaptation for this recently defined plasmid group, which we propose to name "PromA".

RecQ and RecG helicases have distinct roles in maintaining the stability of polypurine·polypyrimidine sequences

DNA triplex structures can block the replication fork and result in double-stranded DNA breaks (DSBs). RecQ and RecG helicases may be important for replication of such sequences as RecQ resolves synthetic triplex DNA structures and RecG mediates replication restart by fork regression. Primer extension on an 88 bp triplex-forming polypurine·polypyrimidine (Pu·Py) tract from the PKD1 gene demonstrated that RecQ, but not RecG, facilitated primer extension by T7 DNA polymerase. A high-throughput, dual plasmid screening system using isogenic bacterial lines deficient in RecG, RecQ, or both, revealed that RecQ deficiency increased mutation to sequence flanking this 88 bp tract by eight to ten-fold. Although RecG facilitated small deletions in an 88 bp mirror repeat-containing sequence, it was absolutely required to maintain a 2.5 kb Pu·Py tract containing multiple mirror repeats. These results support a two-tiered model where RecQ facilitates fork progression through triplex-forming tracts and, failing processivity, RecG is critical for replication fork restart.

Construction and screening of eukaryotic expression plasmids containing short hairpin RNA targeting at the myeloid cell leukemia-1 gene

Construction and screening of eukaryotic expression plasmids containing short hairpin RNA targeting at the myeloid cell leukemia-1 gene

Prevalent phenotypes and antibiotic resistance in Escherichia coli and Klebsiella pneumoniae at an Indian tertiary care hospital: plasmid-mediated cefoxitin resistance

The beta-lactam antibiotics, in combination with aminoglycosides, are among the most widely prescribed antibiotics. However, because of extensive and unnecessary use, resistance to these drugs continues to increase. In recent years, resistance in the Indian bacterial population has increased markedly, the majority showing complex mechanisms. Due to increased transcontinental movement of the human population, it would be wise to know the prevalence and resistance complexity of these strains, well in advance, in order to formulate a policy for empirical therapy. One hundred and eighty-one isolates of Escherichia coli and 61 isolates of Klebsiella pneumoniae obtained from 2655 non-repeat samples of pus (912) and urine (1743) were studied, and their resistance rates and patterns were noted. The isolates were analyzed for prevalent aminoglycoside and cephalosporin resistance phenotypes and for the presence of extended spectrum beta-lactamase (ESBL) and AmpC enzymes by spot-inoculation and modified three-dimensional tests developed in our laboratory. Fourteen isolates of E. coli and six of K. pneumoniae, resistant to all of the antibiotics tested, were selected for plasmid screening, curing, and transconjugation experiments, and for comparative evaluation of the double disk synergy test (DDST) and modified three-dimensional test (TDT) for detection of beta-lactamases. Urinary E. coli isolates showed maximum susceptibility to amikacin (57.1%), followed by tobramycin (38.5%) and gentamicin (31.9%). Eighteen (19.8%) isolates were susceptible to cefotaxime, whereas 11 (12.1%) were susceptible to ceftriaxone. The K. pneumoniae isolates from urine samples showed maximum susceptibility to tobramycin (63.6%) followed by amikacin (54.5%). Of the K. pneumoniae isolates, 31.8% were susceptible to cefotaxime and 13.6% were susceptible to ceftriaxone. A more or less similar trend of antibiotic susceptibility was noted in E. coli and K. pneumoniae isolates from pus samples. Twenty-six (14.4%) E. coli and 15 (24.6%) K. pneumoniae isolates were found to be ESBL-producers by NCCLS-ESBL phenotypic confirmatory test. Eighteen (9.9%) E. coli and 19 (31.1%) K. pneumoniae isolates were found to be AmpC enzyme-producers by our modified TDT. The simultaneous occurrence of ESBL and AmpC enzymes was noted in 7.7% and 9.8% isolates of E. coli and K. pneumoniae, respectively. The prevalence of multidrug-resistant bacterial isolates is quite high in our bacterial population. On comparative evaluation of DDST and TDT in resistant isolates, TDT was found to be the better method, detecting ESBLs in 80% of isolates compared to 15% with DDST. A 19.9-kb plasmid was consistently present in all the screened isolates of E. coli and K. pneumoniae, and was inferred to encode cefoxitin and tetracycline resistance based on curing and transconjugation experiments.

Textile effluent biodegradation potentials of textile effluent-adapted and non-adapted bacteria

Environmental pollution has been recognized as one of the major problems of the modern world. The increasing demand for water and the dwindling supply has made the treatment and reuse of industrial effluents an attractive option. Textile effluents are of concern because they colour the drains and ultimately the water bodies. They also diminish the water quality. The ability of microorganisms to degrade and metabolize a wide variety of compounds has been recognized and exploited in various biotreatment processes. This study investigated the potential of bacteria isolated from textile industries wastewater and drains (textile effluent adapted bacteria) and isolates from a municipal landfill (effluent non-adapted bacteria). We discovered effluent adapted strains of Acinetobacter, Bacillus and Legionella with potentials for colour removal and strains of Acinetobacter, Bacillus and Pseudomonas with chemical oxygen demand (COD) removal activities. Only strains of Bacillus with potentials for use in colour and COD removal were isolated from the landfill. Plasmid screening did not reveal the presence of plasmids in the isolates. Thus the involvement of extra-chromosomal genes is not suggested. In conclusion, as a preliminary step in the development of textile effluent biotreatment using indigenous microbes, we have discovered some strains with potency to decolourize and/or remove COD.

Screening for plasmids among human bifidobacteria species: Sequencing and analysis of pBC1 from Bifidobacterium catenulatum L48

Analysis of 72 bifidobacterial isolates for plasmid DNA identified six different plasmid profiles, two profiles consisted of a single plasmid and four contained at least two. A plasmid identified in a Bifidobacterium catenulatum strain (pBC1) was chosen for further characterization based on its small size and stability. The plasmid was shown to be a circular molecule of 2540 base pairs with an overall G + C content of 64%. At the putative origin of replication a direct repeat of 24 nucleotides repeated three and a half times was observed, as well as five inverted repeats, which resembled the organization of theta-type replicating plasmids. Three open reading frames encoding peptides larger than 50 amino acids were also identified: repB, encoding a replicase of 315 amino acids, a transcriptionally coupled gene ( orfX-like), similar to the orfX of some theta-replicating lactococcal plasmids, and copG-like in the complementary strand, which showed a conserved domain present in proteins of the CopG family. Comparison of the deduced RepB protein of pBC1 to other replication proteins in databases, identified pMB1 from Bifidobacterium longum as its closest relative (81% amino acid identity). The pBC1 replicon proved to be functional in several Bifidobacterium species, including B. animalis, B. longum, and B. pseudocatenulatum. Hybridization experiments showed the replicon was uncommon among bifidobacteria. The relative copy number of pBC1 was estimated to be 30.9 ± 4.62 by quantitative real-time polymerase chain reaction.

Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome

This study describes a novel helicase-mediated isothermal DNA amplification method that exponentially amplifies circular DNAs. The circular helicase-dependent amplification (cHDA) system is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease-deficient T7 DNA polymerase (T7 Sequenase) and the T7 Gp2.5 single-stranded DNA-binding (SSB) protein. After the duplex DNA template is unwound by the T7 helicase, specific primers anneal to the separated DNA strands and T7 Sequenase extends the 3′ end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template. The cHDA reaction can be carried out at one temperature (25°C) for the entire process and can achieve up to 10 000-fold amplification. Amplification can be performed using purified plasmid DNA or a crude cell lysate and can amplify inserts as large as 10 kb. Following a cHDA reaction, the amplified products can be used directly for sequencing and restriction enzyme digestion without further purification. By utilizing the helicase enzyme, circular DNA samples can be simultaneously screened and amplified at one constant temperature in one easy step.

Shuttle vectors for genetic manipulations inUstilago maydis

Shuttle vectors with new or improved features were constructed to enable facile genetic manipulations in the plant pathogen Ustilago maydis. Sets of plasmids selectable in media containing geneticin, carboxin, nourseothricin, or hygromycin, able to replicate autonomously, to transform U. maydis by integration, and to express foreign genes under control of the homologous glyceraldehyde-3-phosphate dehydrogenase promoter, were built upon a common pUC19 vector backbone. This permits a large number of choices for a cloning site, blue/white screening for recombinant plasmids, rapid transfer of a cloned DNA fragment between plasmids, and choice of several dominant drug-resistance markers for selection in U. maydis.

Rapid extraction of plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi.

Hyperthermophilic archaea, specifically Pyrococcus spp., are the target of current efforts in developing heterologous expression systems. However, the published plasmid purification and plasmid screening protocols are long and tedious. We describe a fast, simple protocol for plasmid purification from Pyrococcus spp. developed while extracting the plasmid pGT5 from Pyrococcus abyssi cells. The protocol is modified from the procedures for commercial plasmid minipreps and is completed in about 20 min. The DNA is easily digested by restriction enzymes and can be used in sequencing reactions without additional purification.

New Cloning Vectors with Temperature-Sensitive Replication

A series of cloning vectors with conditional, temperature-sensitive replication that are selectable with ampicillin, chloramphenicol, and kanamycin has been constructed. These vectors are derivatives of a pSC101 mutant that can replicate only at low temperatures. The cloning vectors carry a number of unique restriction sites and provide for screening of recombinant plasmids by α complementation. These vectors have proven useful for a variety of applications where conditional replication of a recombinant plasmid is desired.

Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression.

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria.

Easy screening of recombinant plasmids

Easy screening of recombinant plasmids

Expression, Purification, and Characterization of an Active RNase H Domain of the Hepatitis B Viral Polymerase

The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate. This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product. The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities. Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems. The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis. We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification. The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture. This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component. The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.

Screening for recombinant plasmids in yeast colonies of the two-hybrid system using PCR.

Screening for recombinant plasmids in yeast colonies of the two-hybrid system using PCR.

Molecular epidemiology for local outbreaks of methicillin resistantStaphylococcus aureus (MRSA)

Subtyping isolates may be useful for epidemiological studies of methicillin-resistant-Staphylococcus aureus (MRSA) outbreaks. Among subtyping methods, DNA-based techniques have been applied very effectively for this purpose. An outbreak of MRSA infections took place in one hospital in Barcelona early during 1991. From the beginning of the outbreak to December 92, 70 MRSA isolates from different patients and sources were collected. All strains were evaluated by restriction endonuclease analysis of plasmid DNA (REAP) and macrorestriction endonuclease analysis of genomic DNA using Sma I and pulsed-field-gel-electrophoresis (PFGE). Plasmid screening and REAP using Hind III demonstrated two plasmid subtypes: subtype A showing a large plasmid, and subtype B showing the same large plasmid plus a smaller one. Subtypes A and B corresponded to the more recent and older isolates, respectively, suggesting the loss of the small plasmid during the epidemic. PFGE using Sma I displayed two closely related profiles (PFGE subtype A and A'; CS = 0.90). These subtypes were different from those subtypes exhibited from 4 methicillin-susceptible-Staphylococcus aureus (MSSA) isolates from the same hospital and from 2 epidemiologically unrelated MRSA isolates. Almost all isolates showing PFGE subtype A preceded those isolates showing PFGE subtype A'. This fact and the similarity between both subtypes suggested minor chromosomal DNA rearrangement during the outbreak from a unique strain. While PFGE using Sma I is a useful tool in evaluation of clonal dissemination, our data suggest epidemic or local outbreaks may need several methods to best delineate the source and spread of MRSA strains. The reproducibility and discriminatory power of REAP makes it a useful adjunct in this context.

Transformation of Acinetobacter calcoaceticus RAG-1 by electroporation

A method is described for transforming Acinetobacter calcoaceticus RAG-1 with a transposon-delivery plasmid, pLOFPttKm, and the broad-host-range cloning vector pKT230. Kanamycin-resistant transformants isolated following electroporation with pLOFPttKm were shown to have lost the plasmid, and contained the transposon mini-Tn10PttKm in at least five unique, single sites. Transformation with pKT230 was confirmed by plasmid screening and acquisition of the kanamycin-resistant, streptomycin-resistant phenotype. By optimizing conditions of electric field strength and pulse length for electroporation, we obtained a maximum efficiency of 4.36 × 102/(μg DNA∙109 cells) for insertion of the mini-Tn10PttKm transposon, and 9.88 × 104/(μg DNA∙109 cells) for transformation with plasmid pKT230. Our results indicate that electroporation is a suitable method for introducing transposon and cloning vectors into A. calcoaceticus RAG-1, and possibly other Acinetobacter spp. The availability of a transformation system should enhance the molecular genetic and physiological analysis of these bacteria, particularly the vast majority of strains that are not competent for transformation with DNA.Key words: electroporation, Acinetobacter, transformation, plasmid, transposon.

Screening of plasmids from wastewater bacteria

Research on the plasmids in wastewater bacteria was carried out. From the results of comparative studies on plasmid screening methods, the method of Kado and Liu was considered to be most suitable for detecting plasmids in wastewater bacteria among the five methods tested. Forty five antibiotic resistant bacteria and 10 dominant bacteria were isolated from wastewater samples, and their plasmid contents were examined by using Kado and Liu's method with minor modifications. In total, 31 plasmids were detected in 14 of 45 antibiotic resistant strains, with 10 strains carrying multiple (2–4) plasmids. Their sizes were in the range of 1.8-168 kb, and plasmids with sizes 20–30 kb and less than 10 kb were dominant (29 and 26%, respectively). Several large plasmids detected in antibiotic resistant strains seem to be conjugative. On the other hand, 4 of 10 dominant strains had multiple (4–9) plasmids, and 63% of them were less than 10 kb. So it appeared that plasmids were ubiquitous in wastewater bacterial populations.