Extraction Of Chromosomal DNA Research Articles

Isolation and Identification Bacteria Pseudomonas aeruginosa from adelt patients infected of otitis media and Curing of some Antibiotic Resistance and found content of G+C%

Sixteen isolate of Pseudomonas aeruginosa were collected from patients infected of otitis mediafor adelt people and diagnosed by biochemical teset and API20E these isolate tested for their resistance to Ampicillin, Amoxillin, Chloramphenicol, Trimethoprime, Tetracyclin, Cephotaxim, Amikacin, Gentamycin, Rifampicin and ciproflaxin. For the removal of the resistance against the antibiotics in our isolates plasmid curing experiment was performed by using physical agent (elevated temperature). The results indicated that high temperature (47°C) was efficient to cure all the plasmids, and some isolates became sensitive to antibiotics. The extraction of chromosomal DNA was done to estimate the mol% of G+C of DNA content to all isolates the results the results showed that it was a taxonomic importance and it was at range (66.1- 67.3%).

Plasmid Profile and Enterobacterial Repetitive Intergenic Consensus-PCR Characterization of Salmonella Infantis Isolates Recovered From Poultry Sources

Background: Salmonella is known as one of the most important bacterial agents infecting both humans and animals. Salmonella Infantis has been reported as one of the 15 most prevalent serovars worldwide. Despite its clinical importance, there is little information on the molecular characteristics of S. Infantis in Iran. Objectives: This study was conducted to characterize S. Infantis isolates collected from poultry sources in the last decade. The isolates were typified by plasmid profile and enterobacterial repetitive intergenic consensus (ERIC-PCR). Methods: Forty S. Infantis isolates from poultry sources were subjected to plasmid profile and ERIC-PCR characterization. We used a commercial plasmid extraction kit to extract and purify plasmid DNA which then was separated by gel electrophoresis and viewed under a UV transilluminator. For ERIC-PCR, a commercial bacterial chromosomal DNA extraction kit was used. In this study, we chose ERIC2 primer for the ERIC-PCR test. Results: The plasmid profile revealed that 35% of isolates did not contain any plasmids, but the rest (65%) carried a variable number of plasmids with different molecular weights. Six plasmid profiles were found among 40 S. Infantis isolates. Using ERIC2 primer, 7 profiles were found among 40 S. Infantis isolates in ERIC-PCR. Bands with molecular weights ranging from 400 to 3000 bp were observed. Conclusion: This study provided some genetic data on S. Infantis isolates recovered from poultry sources, and these data can be used for a broader epidemiological study nationwide. These findings showed that although plasmid and ERIC profiles are valuable in epidemiological studies, they have some limitations, too.

Laboratory diagnosis of urinary tract infections in patients with resistance genes towards antibiotics

Escherichia coli are gram-negative bacteria that cause urinary tract infections (UTIs). UTIs have affected a significant percentage of humans yearly due to bacterial infection. Our study aims to determine the prevalence of resistance genes in E. coli towards sulfamethoxazole. This study included (490) patients with UTIs, and the urine samples were cultured on media. The patients were admitted to the Medical City in Baghdad to treat UTIs. 116 E.coli isolates were isolated from urine specimens, 35 isolates of them were resistant to trimethoprim/sulfamethoxazole, and 81 isolates were sensitive to trimethoprim/sulfamethoxazole; the E. coli isolates were submitted to multiplex PCR to detection some resistance genes (Sul1, sul2) after detected the isolates by PCR depending on 16S rRNA. Our study showed that identified E. coli was (91-99%) depending on the number of the examined samples by the Vitek 2 system. The molecular study included extraction of chromosomal DNA from (53) E. coli isolates; 35 samples were taken resistant to antibiotics, while from the total of 81 sensitive isolates, only 18 sensitive samples were taken from that are the most sensitive to Timethprime/sulfamethoxazole, then identification by 16S rRNA gene. Detection of Sulfonamides resistance genes included sul1 and sul2. The results showed the 16S rRNA gene identification found in all E. coli isolates and the detection of antibiotic resistance genes. The resistant isolates with the Sul1 gene prevalence were 11(31%), while the sensitive isolates with Sul1gene were 1(6%). Moreover, the resisted isolates with Sul2 gene prevalence was 8(23%), while the sensitive isolates with the Sul1 gene were 0(0%). The numbers of the resistant isolates were (11) and (8) that carry the Sul1 gene and Sul2 gene, respectively, while the numbers of the sensitive isolates were (1) and (0), respectively. We can conclude that a high percentage of Sul1 gene and Sul2 genes in E. coil isolated from UTIs were high. Keywords. UTI, Sul1, Sul2, resistant gene, trimethoprim-sulfamethoxazole

Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis.

To construct a vacA-knockout Helicobacter pylori mutant strain, whose only difference from the wild strain is its disrupted vacA gene. A clone containing kanamycin resistance gene used for homologous recombination was constructed in a directional cloning procedure into pBluescript II SK, and then transformed into vacA+ H pylori by electroporation. Colonies growing on the selective media containing kanamycin were harvested for chromosomal DNA extraction, and the allelic exchange was determined by polymerase chain reactions and sequencing. Loss of vacuolating activity of the vacA-knockout strain was confirmed by examining the gastric cells co-cultured with cell-free supernatants from H pylori wild strain or the mutant. We constructed a vacA-knockout strain of H pylori through direct mutagenesis, which creates an important precondition for the future research on virulence comparison with gene expression analysis.

Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR.

Colony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3% DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1x 10(5) viable cells for Ralstonia eutropha. Nineteen PHA-positive bacteria were used to evaluate this PCR protocol; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHA-positive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible.

Several different clones present during the penetration phase of resistant Streptococcus pneumoniae in the city of Malmö, Sweden.

A rapid increase in the prevalence of Streptococcus pneumoniae with reduced sensitivity to penicillin (MIC > or = 0.12 microgram/mL) was noted among clinical isolates during a 15-month period in 1994-1995 in the city of Malmö, Sweden. All first-time clinical isolates (n = 178) were consecutively collected and investigated for genetic relatedness with BOX-A PCR and arbitrarily primed (AP) PCR. An improved method for chromosomal DNA extraction and the use of three reliable discriminatory primers for AP-PCR of S. pneumoniae are described. Using molecular fingerprinting, 30 different genotypes were discerned among the 178 isolates. The majority (87%) of isolates belonged to serogroups 6, 9, 15, 19, and 23. Resistance patterns and serogrouping indicated the presence of at least three major phenotypic clones. DNA fingerprinting in conjunction with minimum inhibitory concentration (MIC) values, resistance patterns, and epidemiological data confirmed the existence of two clones belonging to serogroups 6 and 9. Of the 178 isolates, 82% were from children of preschool age, most of whom attended day-care centers. We conclude that abundant S. pneumoniae strains with MICs > or = 0.12 microgram/mL for penicillin were present in the city of Malmö during the study period. At least two genetically discrete clones causing clinical illness were identified, and attendance at day-care centers may be a major factor in the spread of these strains.

Improvement of chromosomal DNA extraction from different yeast species by analysis of single preparation steps.

A modified procedure is proposed for chromosomal DNA extraction based on a cell-wall lytic enzyme never applied before in pulsed field gel electrophoresis. Protoplasting efficiency is retained under very challenging conditions for enzyme activity, such as those required for non-Saccharomyces yeasts often characterized by cell walls highly resistant to lysis.

Comparison of epidemic neisseria meningitidis strains by dna restriction endonuclease profile analysis

Meningococcal disease (MD) in Western Australia prior to 1987 was caused by endemic, predominantly serogroup B Neisseria meningitidis strains. In 1987/S3 an epidemic of serogroup A MD occurred in remote outback Aboriginal communities in Western Australia. Comparable epidemiologic features of this epidemic with overseas epidemics suggested spread of a single clone of serogroup A Neisseria meningitidis . DNA analysis of serogroup A strains isolated from MD cases admitted to Princess Margaret Hospital for Children in 1987/88 was undertaken. Chromosomal DNA extraction and restriction endonuclease profiles obtained with EcoRI and Hind III demonstrate that these serogroup A strains are clonal. This indicates that the molecular epidemiology of epidemic serogroup A MD in Western Australia is similar to recent experience in Saudi Arabia, Sudan, Chad and Ethiopia, where spread of single closely related clones has been iesponsible for pandemic waves of MD in the past decade.

In situ digestion of satellite DNA of Scilla siberica

Restriction endonucleases have been used to digest DNA in fixed metaphase chromosomes of animal species. However, constitutive C-heterochromatin of plant species is resistant to these enzymes suggesting that the special structural organization of plant C-bands is an impediment to the activity of restriction endonucleases. In order to test this hypothesis, we have chosen the species Scilla siberica, whose purified satellite DNA, localised at the heterochromatic regions, is extensively digested by HaeIII. In situ treatment with HaeIII alone does not produce significant digestion of heterochromatin, but subsequent treatment with proteinase K results in extensive digestion of heterochromatic regions producing unstained gaps. These results indicate that HaeIII is able to access and cut chromosomal DNA from C-bands, but the DNA fragments remain attached to chromosomal proteins that characterize the complex structure of heterochromatin in this species. Although there are no reasons to suppose that accessibility of chromosomal DNA of S. siberica to restriction enzymes can be impeded, it would be reasonable to think from our results that some special features of heterochromatin organization in plants contribute to the formation of a complex structure that makes chromosomal DNA extraction impossible.

The identification, typing and fingerprinting of Salmonella: laboratory aspects and epidemiological applications

Since the 1930s, traditional methods of strain identification based on serotyping and phage typing have been the foundation of salmonella epidemiology. Although the incidence of diseases such as typhoid and paratyphoid has decreased in recent years, food-poisoning caused by non-typhoidal salmonella strains has now reached epidemic proportions in many countries, despite improvements in sanitation and hygiene. Precise strain identification is an essential prerequisite for epidemiological investigations aimed at combating the spread of these strains and eradicating the sources of infection. Modern methods of genotypic typing, particularly those based on physical characterization of the plasmid content of the organism have already proved invaluable for the identification and differentiation of strains in many outbreaks. These plasmid typing methods are now increasingly used with serotyping and phage typing for many epidemiological investigations. Other methods of genotypic typing, particularly those based on recognition of small differences in chromosome structure, are not yet practical for the examination of large numbers of strains. Nevertheless, improvements in small-scale methods for chromosomal DNA extraction coupled with the increasing use of non-isotopic labels for identification of restriction fragment length polymorphisms may provide a new dimension to Salmonella epidemiology.