Alkaline Lysis Method Research Articles

Evaluation of Genetic Pattern of Gentamicin-resistant Enterococci Isolated from Clinical Samples.

Background and aims Enterococci are among resistant microorganisms to antibiotics and are responsible for some of acute gingival infections. This study aimed at molecular evaluation of gentamicin-resistant enterococcus species and determining the resistance genes. Materials and methods Clinical samples were cultured on BHI medium and enterococci were isolated by specific methods and antibiograms were prepared. DNA was derived from gentamicin-resistant species by alkaline lysis method and replicated by PCR method. Four gentamicin-resistant genes were evaluated by electrophoresis method on agarose gel. Results From 105 Enterococcus faecalis isolates, 36.2% and from 38 Enterococcus faecium isolates, 78.2% were resistant to gentamicin. 60% of these species bore aac (6’)- le – aph(2”)- la gene. ph(2”)- Ib,Ic,Id genes were not seen in any of the evaluated species. Conclusion In gentamicin-resistant species of E. faecalis and E. faecium, the aac (6’)- le – aph(2”)- la gene was identified as the main cause of resistance.

Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Plasmid Fingerprinting Profiles of Chicken-Derived Pathogenic Strains of <i>Escherichia coli</i>

Plasmid DNA was extracted from all 38 strains of chicken Escherichia coli by alkaline lysis method. The agarose gel electrophoresis showed 23 plasmid patterns. The strains from the same source had the same, similar, or quite different plasmid profiles, while those from different sources generally did not have the same plasmid profiles. Therefore, the plasmid profiles can provide basic materials for epidemiological investigation on chicken Escherichia coli at molecular level.

Sequence analysis of plasmid in Klebsiella pneumoniae KF3

Objective To study the structures of the plasmids of Klebsiella pneumoniae KF3 at the genome metagenome level througth with whole plasmid DNA sequencing, to analyze the functional genes carried by plasmid and to identify the correlation of resistance and pathogenicity between the plasmids and the host strains. Methods The alkaline lysis method was used to extract plasmids. We constructed the small insert pUC18 library and the large insert Forsmid library, sequenced and used the Phred / Phrap / Consed package to assemble these sequences and gained a complete sequence. The open reading frame(ORFs) were predicted by the Glimmer software and annotated, analyzed the functions of these genes. Results We successfully constructed the pUC18 library and the Fosmid libraries for the plasmid DNA and obtained three circular double-stranded DNA plasmids: pKF3-70 (69 477 bp), pKF3-90 (91 327 bp) and pKF3-147 ( 147 416 bp). There were drug resistant genes, conjugative transfer genes and mobile DNA elements identified on three plasmids. Conclusion The three plasmids of KF3 could be transferred among different strains. It would lead to the dissemination of the resistant genes. Key words: Plasmids; Drug resistant genes; Conjugative transfer genes; Klebsiella pneumoniae

Subtype prevalence, plasmid profiles and growing fluoroquinolone resistance in Shigella from Kolkata, India (2001-2007): a hospital-based study

Shigellosis is a major public health problem, and increasing antimicrobial resistance has complicated its treatment. We report isolation frequency, plasmid profiles and antimicrobial resistance of Shigella subtypes in Kolkata, India, from a prospective hospital-based study. Fresh stool or rectal swabs were collected from children (<5 years) attending the Diarrhea Treatment Unit of a governmental paediatric referral hospital in Kolkata. Samples were processed following standard methods over a 7-year period from January 2001 to December 2007. Of 4478 samples collected, 516 (11.5%) were positive for Shigella spp. S. flexneri (312; 6.9%) was the most frequently isolated serogroup, ranking before S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) and S. boydii (33, 0.7%). Although 10 subtypes of S. flexneri were identified, the common ones circulating locally were S. flexneri 2a (179), S. flexneri 6 (38) and S. flexneri 3a (36). Knowledge of Shigella subtypes is important for vaccine development. The majority of Shigella isolates (81.0%) were multidrug (two or more antimicrobial classes) resistant and showed high minimum inhibitory concentration (MIC) with commonly used drugs like ampicillin, tetracycline, co-trimoxazole and nalidixic acid. Emergence of fluoroquinolone (FQ)-resistant S. dysenteriae type 1 (100.0%) in 2002-2003 was followed by frequent isolation (>25.0%) of FQ-resistant S. flexneri 2a, and S. flexneri 3a in 2004, which restricted use of fluoroquinolones for treatment. A number of smaller plasmids (<20 kb) with distinct patterns have been observed for several years in predominant subtypes. Long-term surveillance of Shigellae and their antimicrobial resistance are mandatory in endemic areas to formulate treatment policy until any suitable candidate vaccine is available to control the disease.

A developed DNA extraction method for different soil samples

Four DNA extraction methods namely SDS-hyperhaline method (I), modified SDS-hyperhaline method (II), indirect method (III), alkaline lysis method (IV) were evaluated by comparing DNA yield, spectrophotometric quality, genomic integrity and PCR suitability in this paper. The results showed that high DNA yields were obtained by method I, II and IV. However, higher quality of DNA was gained by method III and IV. Based on the results of the Pulsed-Field Gel Electrophoresis (PFGE), the completeness of DNA extracted by method IV was the best. About 6.0 microg DNA can be recovered from 1.0 g soil by method IV which involved to lysis cell by SDS and to precipitate impurities by adding potassium acetate and magnesium chloride Therefore, it is confirmed that method IV is a novel, reliable and versatile method for large-scale DNA extraction involving less purification steps for various soil samples.

Plasmid DNA of high quality purified by activated charcoal

Demand for plasmid DNA of high purity and safety has increased with rapid advances in gene therapy and DNA vaccines in addition to basic DNA study. Using activated charcoal (AC), we have developed protocols for pure plasmid DNA. Plasmid DNA extracted by the alkaline lysis method was inevitably contaminated with nucleotide fragments. Treatment with AC during purification instead of RNase completely removed nucleotide fragments in the final plasmid DNA and the removing capability of AC was dose dependent on AC quantity. Of note is that nucleotide fragments less than 0.4 kbp were effectively removed by AC and purification up to 500 ml was easily achieved. Taken together, inexpensive AC effectively removed the troublesome nucleotide fragments and practically substituted for expensive RNase. The resultant plasmid DNA has enough quality needed for basic DNA study and application.

Epidemiology and antimicrobial resistance of staphylococci isolated from different infectious diseases

A total of 187 isolates from 470 clinical specimens were collected from three hospitals in El-Minia governorate and identified as 132 Staphylococcus aureus strains and 55 coagulase-negative staphylococci (CoNS) strains. Susceptibility of isolates to antimicrobial agents was tested by the agar dilution method. The isolated S. aureus strains showed low resistance to vancomycin (1.5%), amikacin (2.3%) and gatifloxacin (3.8%). Vancomycin was the most effective antibiotic against CoNS. The ampicillin-resistant isolates were tested for β-lactamase production where, 61.7% of S. aureus and 42.9% of CoNS were positive for β-lactamase enzyme. Beta-lactamase producing strains were screened for their plasmid profile using alkaline lysis method. Some of these strains carried at least one plasmid suggesting plasmid-mediated antibiotic resistance. When cells of these strains were exposed to curing agent ethidium bromide, the production of the β-lactamase was lost. Resistance by efflux was studied by a modified fluorometric assay. Addition of uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased norfloxacin accumulation in quinolone resistant S. aureus strains, suggesting endogenous energy-dependent efflux. Combinations of ciprofloxacin with four antimicrobial agents against methicillin resistant S.aureus (MRSA) strains were investigated using decimal assay for additivity (DAA) technique. Synergistic interaction was observed between ciprofloxacin and oxacillin. ciprofloxacin plus cefepime and gentamicin appeared to be additive, while ciprofloxacin plus erythromycin was antagonistic.

Epidemiology and antimicrobial resistance of staphylococci isolated from different infectious diseases.

A total of 187 isolates from 470 clinical specimens were collected from three hospitals in El-Minia governorate and identified as 132 Staphylococcus aureus strains and 55 coagulase-negative staphylococci (CoNS) strains. Susceptibility of isolates to antimicrobial agents was tested by the agar dilution method. The isolated S. aureus strains showed low resistance to vancomycin (1.5%), amikacin (2.3%) and gatifloxacin (3.8%). Vancomycin was the most effective antibiotic against CoNS. The ampicillin-resistant isolates were tested for β-lactamase production where, 61.7% of S. aureus and 42.9% of CoNS were positive for β-lactamase enzyme. Beta-lactamase producing strains were screened for their plasmid profile using alkaline lysis method. Some of these strains carried at least one plasmid suggesting plasmid-mediated antibiotic resistance. When cells of these strains were exposed to curing agent ethidium bromide, the production of the β-lactamase was lost. Resistance by efflux was studied by a modified fluorometric assay. Addition of uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased norfloxacin accumulation in quinolone resistant S. aureus strains, suggesting endogenous energy-dependent efflux. Combinations of ciprofloxacin with four antimicrobial agents against methicillin resistant S.aureus (MRSA) strains were investigated using decimal assay for additivity (DAA) technique. Synergistic interaction was observed between ciprofloxacin and oxacillin. ciprofloxacin plus cefepime and gentamicin appeared to be additive, while ciprofloxacin plus erythromycin was antagonistic.

Detect of P53,bax and caspase 3 genes expression after optic nerve injury in rats with SYBR green I fluorescence quantitative PCR

Objective Previous study showed that the histopathological basis of visual function damage caused by optical nerve injury is apoptosis of retinal ganglion cells（RGCs）. This procedure is regulated by P53 ,bax and caspase 3 genes. Present study aimed to observe the expression of bax,P53 and caspase 3 mRNA in RGCs after traumatic optic nerve damage in the rats by SYBR green I fluorescence quantitative PCR method. Methods The animal model of optic nerve injury was established in the right eyes of 56 adult Wistar rats by a fluid percussion brain injury device （FPI）. Animal were killed on days 1,3,5,7,9,14,28 days separately after injury. Other 16 Wistar rats were divided into normal control group and sham operation group. The total RNA was isolated from rat fresh retina tissue by Trizol method and was treated by reverse transcription to eDNA using Oligo（dt） 18 as primer and then amplified. The target fragments of bax,P53 and easpase 3 cDNA were linked with carrier pTZ57 R/T to construct recombined plasmids which were transformated to E. coli DH5α by T/A clone method. Recombined plasmids were extracted with alkaline lysis method and the plasmids were selected in white colonies by ampicillin screening,EcoR I restrictive enzyme analysis, and their specificity was evaluated using DNA sequencing. The standard curves were created by plasmid DNA and the precise expression level of target genes in samples were determined using software. The results were expressed as the ratios of target gene mRNA to GAPDH mRNA. Results The standard curve drawn by pTZ57R/T and target gene presented a good linear tendency with the higher sensitivity and specificity. The expression of P53 and bax mRNA began to inerease on the third day after the injury of optic nerve and peaked on the fifth day and started to decline on the seventh day. The expression of caspase 3 mRNA increased from the fifth day through the ninth days after injury and declined on the fourteenth day. The significant differences were found in the expression of P53, bax and easpase 3 between model group and control group （P 〈 0.05 ）. Conclusion The pro-apoptotie protein P53 ,bax and easpase 3 play an important role in RGCs apoptosis. Key words: SYBR green I method; fluorescence quantitative polymerase chain reaction; T/A clone method; traumatic optic nerve injury; P53; bax; caspase 3;

Comparative Analysis Of Antibiotic Resistance And R-Plasmids Of Staphylococcus aureus Isolates From Human And Dog Samples

Bacterial resistance to antibiotics constitutes a major cause of failure in the treatment of bacterial infections. The genetic exchange of plasmids containing antibiotic resistant determinants between bacteria is believed to play a critical role in the evolution of antibiotics resistant bacteria and this has been shown in S. aureus. This study was therefore carried out to investigate the nature of plasmids that determine antibiotic resistance in Staphylococcus aureus isolates from man and animal. Thirty multiply drug resistant S. aureus isolates from a total of 147 apparently healthy humans and dogs, as well as from clinical cases were determined by antibiotic susceptibility test using the standard disc agar diffusion method. Plasmid isolation was carried out by the alkaline lysis method of Birnboim and Dolly. Electrophoresis as well as the transformation experiment was done. The result showed that no particular sensitivity pattern or plasmid profile can be ascribed to either human or animal sources of isolates. Two isolates from a domestic dog and its owner (human) were observed to have identical plasmid profile and almost the same antibiogram. 23.130 kbp and 25.119 kbp plasmids that were responsible for amoxycilin resistance were transferred. In conclusion, the genetic basis of antibiotic resistance by S. aureus in our locality was found to be partly plasmid mediated. Plasmid analysis, in conjunction with the antibiogram is valuable in differentiating multiple resistant S. aureus. Furthermore, domestic pet animals were found to be reservoirs and potential risk factors in the transfer of multiply antibiotic resistant S. aureus and their R-plasmids to antibiotic susceptible S. aureus and other bacteria.

Analysis of Antimicrobial Resistance and Plasmid Profiles inSalmonellaSerovars Associated with Tropical Seafood of India

A total of 256 Salmonella strains consisting of 29 Salmonella serovars isolated from seafood of Cochin (India) were analyzed for resistance to antimicrobials commonly used in human and veterinary medicines as therapeutic agents. The 10 most predominant Salmonella serovars in seafood were also characterized for presence of plasmids using the alkaline lysis method. Antimicrobial susceptibility studies highlighted a comparatively high resistance in Salmonella isolates to sulfamethizol and carbenicillin, and moderate resistance to nalidixic acid and oxytetracycline. Nevertheless, antimicrobial resistance was not observed against ampicillin, ciprofloxacin, chloramphenicol, gentamicin, and kanamycin in different Salmonella serovars. Fifty percent of the Salmonella isolates, comprising 16 Salmonella serovars, were resistant to sulfamethizol followed by 39% resistant to carbenicillin and 14% resistant to oxytetracycline. Multidrug resistance was detected in 39.4%, 14.4%, 12.1%, and 1.5% of Salmonella isolates towards two drugs (sulfamethizol and carbenicillin), three drugs (sulfamethizol, carbenicillin, and oxytetracycline), four drugs (sulfamethizol, carbenicillin, oxytetracycline, and nalidixic acid), and five drugs (sulfamethizol, carbenicillin, oxytetracycline, nalidixic acid, and streptomycin), respectively. Plasmid profiling highlighted the presence of nine plasmid profiles in Salmonella serovars and plasmids that were not detected in Salmonella enterica subsp. enterica Weltevreden, Salmonella Rissen, Salmonella Bareilly, Salmonella Irumu, Salmonella Ohio, Salmonella Oslo, and Salmonella Typhi isolated from seafood.

Characterization of Extended-Spectrum β-Lactamases in Enterobacteriaceae Causing Nosocomial Infections in a Zagreb University Hospital

The bacteria producing extended-spectrum β-lactamases (ESBLs) are increasingly reported. Production of ESBLs by Gram-negative bacteria is the major mechanism of resistance to oxymino-cephalosporins and aztreonam. The aim of the present study was to characterize ESBLs produced by Enterobacteriaceae, collected during 2003-2005 in a University Hospital in Zagreb, and to determine the risk factors associated with nosocomial infections due to them. 76 isolates of Enterobacteriaceae were included in the study. Antibiotic susceptibility testing was performed by disk-diffusion and broth microdilution method according to CLSI. β-lactamases were characterized by PCR and sequencing of blaESBL genes. Plasmids were extracted by alkaline lysis method and digested with EcoRI enzyme. Most of the strains displayed CAZ phenotype meaning a higher level of resistance to ceftazidime compared to cefotaxime and ceftriaxone. 50 strains produced SHV-ESBL, 28 tem and 8 CTX-M β-lactamase. Sequencing of blaSHV genes from representative strains revealed SHV-5 β-lactamase in 6 strains whereas sequencing of blaCTX-M genes identified CTX-M-3 β-lactamase in 3 and CTX-M-15 in 5 strains. Strains were assigned to groups from A to F according to plasmid fingerprinting. the spread of SHV-5-producing strains throughout the hospital units could be due to selective pressure of ceftazidime which is widely prescribed in our hospital thus favoring survival of strains possessing a mutation at the Ambler position 240 responsible for ceftazidime and aztreonam resistance.

Emergence of Clonally Related SHV-5 ESBL-producing Escherichia coli Strains in the Community

Background: Extended-spectrum β -lactamase (ESBL)-producing Escherichia coli is an emergent cause of urinary tract infections in nonhospitalized patients in different countries. The aim of this study was the molecular characterization of ESBL-producing E. coli strains isolated from urine of outpatients in Zagreb region. Materials and Methods: During the five-month study period a total of 2, 451 E. coli strains were isolated from urine of nonhospitalized patients with significant bacteriuria. ESBL production was detected by double-disk diffusion technique and by >3-dilution reduction in the minimal inhibitory concentration of ceftazidime in the presence of clavulanate. A total of 39 ESBL-producing E. coli strains (1.59%) were collected and characterised. Molecular relatedness between the strains was analyzed by pulsed-field gel electrophoresis (PFGE) and isolates were considered to be genetically related if the Dice coefficient correlation was >80%.  -lactamases were characterized by isoelectric focusing, substrate profile determination, polymerase chain reaction and sequencing of blaSHV genes. PCR products were subjected to PCR Nhe test to distinguish between SHV-1 and SHV-ESBL. Plasmids were extracted by alkaline lysis method and digested with EcoR1 enzyme. Results: Chromosomal DNA analysis by PFGE exhibited a great genomic similarity among ESBL strains. The Dice coefficient of similarity for 25 strains was ≥ 83.49%. All strains produced a  -lactamase with the isoelectric point of 8.2. Enzymes produced by the strains antagonized the activity of the disks containing ceftazidime, cefotaxime, ceftriaxone and aztreonam but not cefoxitin and imipenem. All strains yielded an amplicon with primers specific for SHV β -lactamases. PCR Nhe test demonstrated SHV-ESBL in all tested strains. Fifteen strains possessed an additional TEM β -lactamase. No CTX-M β -lactamases was found. Based on sequencing of blaSHV genes enzymes of five strains were identified as SHV-5  -lactamase which conferred on the producing isolates high level of ceftazidime and aztreonam resistance. Mutations at the Ambler positions 238 and 240 were found in all gene sequences. The blaSHV genes were encoded on related plasmids which also carried resistance genes for aminoglycosides. Plasmids were assigned to nine fingerprinting patterns (A-F). Conclusion: The study demonstrated community-associated emergence of clonally related SHV-5 β -lactamase– producing E. coli strains. Other studies on ESBLs in the outpatient populations reported CTX-M ESBLs to be most prevalent in the community setting. The fact that the strains were clonally related, that produced the same type of ESBL and that the high proportion of strains were resistant to aminoglycosides which are not prescribed outside of hospitals, could indicate the possible food origin of ESBL E. coli strains.

Location and PCR analysis of catabolic genes in a novel Streptomyces sp. DUT_AHX capable of degrading nitrobenzene

A novel strain of Streptomyces sp. DUT_AHX was isolated from sludge contaminated with nitrobenzene and identified on the basis of physiological and biochemical tests and 16S ribosomal DNA (rDNA) sequence analysis. The optimal degradation conditions were as follows: temperature 30 degrees C, pH 7.0-8.0, shaking speed 150-180 r/min, and inocula 10% (V/V). The strain, which possessed a partial reductive pathway with the release of ammonia, was also able to grow on mineral salts basal (MSB) medium plates with 2-aminophenol, phenol, or toluene as the sole carbon source. Furthermore, the enzyme activity tests showed crude extracts of nitrobenzene-grown DUTAHX contained 2-aminophenol 1,6-dioxygenase activity. The 17-kb plasmid was isolated by the modified alkaline lysis method and was further cured by sodium dodecyl sulphate (SDS) together with 37 degrees C. As a result, the cured derivative strain DUTAHX-4 lost the 2-aminophenol 1,6-dioxygenase activity. The results suggested that the catabolic genes encoding the nitrobenzene-degrading enzymes were plasmid-associated. Moreover, the plasmid DNA was amplified with degenerate primers by touchdown PCR and an expected size fragment (471 bp) was generated. The Blast results revealed that the gene encoding a 157 amino acid polypeptide was 39%-76% identical to YHS domain protein. The further examination of the plasmid would demonstrate the molecular basis of nitrobenzene catabolism in Streptomyces, such as regulation and genetic organization of the catabolic genes.

Plasmid profile analysis in identification of epidemic strains of Salmonella enterica serovar Enteritidis

As illness caused by Sallmonella enterica serovar Enteritidis (S. Enteritidis) occurs not only as sporadic cases but as outbreaks, to reveal the source and routes of spreading of infection it is necessary to identify epidemic strain by the use of some typing methods. To determine whether plasmid profile analysis, as genotyping method, could be applied for the investigation of epidemic strains, isolates of S. Enteritidis, recovered from patient's stools and food associated with outbreaks and those isolated from sporadic cases of diarrhea, were investigated. Investigation of antibiotic resistance was performed by Kirby-Bauer disc-diffusion method. Isolation of plasmid DNA was carried out by Birnboim and Dolly alkaline lysis method, modified by Ish-Horovitz. Out of 276 izolates of S. Enteritidis 94 were isolated from patient's stools and food associated with outbreaks and 182 were isolated from sporadic cases of diarrhea. The presence of 12 plasmid profiles was established. An average correlation degree of plasmid profiles between the strains was 0.84, that implies high degree of similarity of plasmid profiles of epidemic and non epidemic strains isolated at our geographic region for the given period of time. The strains of S. Enteritidis, isolated in outbreaks of enterocolitis as well as from spordic cases of diarrhea in the same period of time and at the same area, frequently exhibit the same plasmid profile characterized by a single plasmid of 38 MDa. Therefore, in most cases plasmid profile analysis is not valuable in the identification of epidemic strains of S. Enteritidis. However, for this purpose plasmid profile analysis could be used when drug-resistant strains of S. Enteritidis are isolated, as they often possess additional resistant plasmids what increases discrimination power of this method.

A Simple, Rapid Method for Extracting Large Plasmid DNA from Bacteria

We are studying the lateral transfer of transmissible antibiotic resistance plasmids among stream bacteria impacted by fecal runoff from poultry and cattle. Such plasmids are typically large (ca. 40 – 100 kb) and occur in low copy numbers in the cell and have therefore typically been difficult to isolate and therefore to study. Traditional protocols, based upon variations of the standard alkaline-lysis method, are long (ca. 1 1/2 to 2 days) and difficult. Commercial kits designed for the isolation of Baterial Artificial Chromosomes (BACs) can be used and are an improvement; however, these are expensive and still require hours of sustained effort. We have adapted a method published by Rondon et al. (1999), originally designed for the isolation of BAC DNA, for the rapid isolation of large plasmid DNA. In this method, lysis and alkaline denaturation steps are combined, incubation steps are vastly reduced, proteins are removed via a simple ammonium acetate/chloroform step, and the DNA precipitated using a plyethylene glycol/NaCl step. No ethanol precipitation is required. If additional purification is required, extracted DNA can be further processed through a Qiagen Plasmid Mini or Midi column (Qiagen Inc., Valencia CA). The method is rapid (under 1 hour), easy, very inexpensive and has been reliably used by undergraduate students to isolate large (up to 200 kb) native plasmids from a variety of both Gram-positive and Gram-negative genera including Shigella, Klebsiella, E. coli, Pseudomonas, Bacills, Streptococcus, Staphylococcus, and Enterococcus, as well as BACs from E. coli. The protocol is simple and reliable enough to be used for the rapid large-scale visualization of native plasmids and we have used it to visualize and isolate DNA from hundreds of multidrug resistance plasmids exogenously captured from stream sediments, soils, and beach sands.

A Simple, Rapid Method for Extracting Large Plasmid DNA from Bacteria

AbstractWe are studying the lateral transfer of transmissible antibiotic resistance plasmids among stream bacteria impacted by fecal runoff from poultry and cattle. Such plasmids are typically large (ca. 40 – 100 kb) and occur in low copy numbers in the cell and have therefore typically been difficult to isolate and therefore to study. Traditional protocols, based upon variations of the standard alkaline-lysis method, are long (ca. 1 1/2 to 2 days) and difficult. Commercial kits designed for the isolation of Baterial Artificial Chromosomes (BACs) can be used and are an improvement; however, these are expensive and still require hours of sustained effort. We have adapted a method published by Rondon et al. (1999), originally designed for the isolation of BAC DNA, for the rapid isolation of large plasmid DNA. In this method, lysis and alkaline denaturation steps are combined, incubation steps are vastly reduced, proteins are removed via a simple ammonium acetate/chloroform step, and the DNA precipitated using a plyethylene glycol/NaCl step. No ethanol precipitation is required. If additional purification is required, extracted DNA can be further processed through a Qiagen Plasmid Mini or Midi column (Qiagen Inc., Valencia CA). The method is rapid (under 1 hour), easy, very inexpensive and has been reliably used by undergraduate students to isolate large (up to 200 kb) native plasmids from a variety of both Gram-positive and Gram-negative genera including Shigella, Klebsiella, E. coli, Pseudomonas, Bacills, Streptococcus, Staphylococcus, and Enterococcus, as well as BACs from E. coli. The protocol is simple and reliable enough to be used for the rapid large-scale visualization of native plasmids and we have used it to visualize and isolate DNA from hundreds of multidrug resistance plasmids exogenously captured from stream sediments, soils, and beach sands.

Antimicrobial Susceptibility and Plasmid Profiles of &lt;i&gt;Salmonella typhi&lt;/i&gt; isolated in Abuja, Nigeria.

Sixty clinical Salmonella spp. isolates were obtained from various health establishments in Abuja, Nigeria. These isolates were characterized using cultural, biochemical and serological assays. Antimicrobial susceptibility and plasmid analysis were used to determine the presence of plasmid-mediated multi-drug resistance. Each isolate was evaluated for susceptibility to ampicillin, chloramphenicol, gentamicin, tetracycline, cotrimoxazole, nalidixic acid, ciprofloxacin and afloxacin, using disk diffusion technique. Plasmid DNA was extracted by alkaline lysis method and fragments separated by electrophoresis on 1% agarose gel. DNA bands were visualized using ultraviolet light transilluminator and molecular weights determined. Plasmid transfer by mating was also performed using Escherichia coli K-12 as recipient. In total, 36 (60.0%) isolates were confirmed to be Salmonella typhi, of which 6 (16.7%) were fully susceptible to all microbial agents tested, while 7 (19.4%) were resistant to three or more antimicrobials including ampicillin, chloramphenicol, tetracycline, nalidixic acid and cotrimoxazole, categorized as ‘first line' drugs in the treatment of typhoid fever. However, all isolates evaluated were susceptible to ciprofloxacin and ofloxacin. Of the seven multi-drug resistant strains, 6 (85.7%) possessed plasmid sizes of 4.7 – 5.7 kilobase, suggesting that transmissible R-plasmids might be responsible for the drug resistance observed in Salmonella typhi strains in this study. Our findings show that plasmid-bearing multi drug resistant strains account for a reasonable percentage of Salmonella typhi isolates in Abuja, Nigeria. Keywords: Salmonella typhi, Plasmid, Antimicrobial resistance, typhoid fever. Journal of Medical Laboratory Sciences Vol. 14 (2) 2005: pp. 46-53

A RAPID AND SIMPLE METHOD FOR SCREENING LARGE NUMBERS OF RECOMBINANT DNA CLONES*

ABSTRACT Screening recombinant DNA clones from cDNA library or transformation experiments is a regular task in molecular biology research; the routine alkaline extraction procedure is tedious and time consuming (90–120 min for 12 samples), and unsuitable for screening large numbers of clones from these experiments, especially when no blue‐white indicator (α‐complementation of lacZ gene) exists in recombinant clones. Some commercial plasmid preparation kits are more rapid (20–60 min for 12 samples) and simple in plasmid extraction; however, they are costly on a daily basis. Therefore, a simple, rapid and economic method for screening recombinant DNA clones is needed. Here we present an extremely simple, rapid and economic procedure of plasmid DNA preparation for screening recombinant clones from cDNA library or transformation experiments based on a modified alkaline lysis method. In this method, bacterial colonies or cultures were directly lysed in 2% NaOH solution, and then recombinant plasmids in the supernatant were visualized quickly by agarose gel electrophoresis. The entire process, from Escherichia coli colonies or cultures to plasmid DNA, takes only about 12–15 min for 12 samples, which is the quickest method for plasmid preparation in our best knowledge. Hundreds of recombinant clones with no easy screening method can be rapidly identified in several hours using this method.PRACTICAL APPLICATIONSOur approach is extremely rapid and simple to screen large numbers of recombinant clones from bacterial colonies or bacterial cultures of cDNA library or transformation experiments. It is especially suitable for identifying those high copy recombinant vectors without blue‐white color reaction such as pDNR‐LIB, pPICZαA, etc. In addition, this protocol is also suitable for extracting plasmids and screening recombinant DNA clones from bacterial cultures. Our data have indicated that plasmids extracted by our method could meet the criteria for restriction enzyme digestion after neutralization.