Gel Electrophoresis Of Chromosomal DNA Research Articles

Dynamics of Indigenous Lactic Acid Bacteria Populations in Wine Fermentations from La Rioja (Spain) During Three Vintages

Diversity of lactic acid bacteria (LAB) species has been analyzed for three consecutive years (2006, 2007, and 2008) during alcoholic and malolactic fermentations of Tempranillo wine in a winery at La Rioja. The results showed differences in malolactic fermentation duration, and in both diversity of LAB species and diversity of Oenococcus oeni genotypes. O. oeni was shown to be the predominant species (73% of total isolates). Monitoring the different strains of O. oeni using pulsed-field gel electrophoresis of chromosomal DNA digested with SfiI and ApaI allowed detection of a total of 37 distinct genotypes, most of them comprised at least two isolates. Six appeared in more than one vintage, one of them being present in the three studied years. Moreover, four genotypes were indistinct of the strains isolated from the air of this same winery in 2007 vintage. The frequency of participation of each genotype varied from year to year, thus dominant genotypes at oneyear were minority or not present at another year. This suggests that distinct indigenous O. oeni strains are better adapted to the different winery conditions every year. Predominant genotypes that appeared in more than one vintage and lead to quality wines with low histamine contents could be considered as interesting for selecting of new malolactic starter cultures.

Identification, antimicrobial resistance and genotypic characterization of Enterococcus spp. isolated in Porto Alegre, Brazil.

In the past two decades the members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. In the present study, we evaluated the antimicrobial resistance and genotypic characteristics of 203 Enterococcus spp. recovered from different clinical sources from two hospitals in Porto Alegre, Rio Grande do Sul, Brazil. The species were identified by conventional biochemical tests and by an automated system. The genetic diversity of E. faecalis presenting high-level aminoglycoside resistance (HLAR) was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. The E. faecalis was the most frequent specie (93.6%), followed by E. faecium (4.4%). The antimicrobial resistance profile was: 2.5% to ampicillin, 0.5% to vancomycin, 0.5% teicoplanin, 33% to chloramphenicol, 2% to nitrofurantoin, 66.1% to erythromycin, 66.5% to tetracycline, 24.6% to rifampicin, 30% to ciprofloxacin and 87.2% to quinupristin-dalfopristin. A total of 10.3% of the isolates proved to be HLAR to both gentamicin and streptomycin (HLR-ST/GE), with 23.6% resistant only to gentamicin (HLR-GE) and 37.4% only to streptomycin (HLR-ST). One predominant clonal group was found among E. faecalis HLR-GE/ST. The prevalence of resistance among beta-lactam antibiotics and glycopeptides was very low. However, in this study there was an increased number of HLR Enterococcus which may be spreading intra and inter-hospital.

Genetic and phenotypic diversity of Bifidobacterium thermacidophilum fecal isolates from newborns

This study was undertaken to genetically identify and phenotypically characterize 14 bifidobacteria isolated from 20 breast-fed newborns. These isolates showed 98%-99% similarity to Bifidobacterium thermacidophilum subsp. suis based on 16S rDNA. Further analysis by pulsed-field gel electrophoresis of chromosomal DNA digested with XbaI revealed 4 distinct restriction patterns. The predominant pattern, shared by 8 (57%) isolates, produced a macro-restriction profile with about 13 large fragments ranging in size from >242.5 to 23.1 kb, whereas the other 6 displayed 3 distinct restriction profiles all characterized by more micro- than macro-restriction, with fragments ranging in size from 97 to 9.4 kb. Phenotypic characteristics, including carbohydrate fermentation profile, maximal growth temperature, and antibiotic susceptibility, varied widely even among strains showing the same restriction profile. The presence of B. thermacidophilum in stools of newborn infants may indicate the potential of these bacteria for aiding the development of the intestinal ecosystem.

Vancomycin-Tolerant Streptococcus pneumoniae in Korea

A nationwide surveillance study was undertaken to monitor antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in Korea, with a special focus on vancomycin tolerance. For the 6-month period from March to August 2002, clinical isolates of S. pneumoniae were collected from 11 university hospitals and 1 reference laboratory. One-hundred eighty-eight isolates were measured for lysis rates after exposure to vancomycin for 4 h. Two vancomycin-tolerant S. pneumoniae (VTSP) strains, S3 and H8, were isolated from sputum cultures of two patients, who had stayed in intensive-care units of different hospitals with long-term antibiotic therapy and were not treated for pneumococcal pneumonia. The penicillin, cefotaxime, and vancomycin MICs for S3 were 8 microg/ml, >16 microg/ml, and 0.5 microg/ml, and those for H8 were 2 microg/ml, 2 microg/ml, and 0.5 microg/ml, respectively. While S3 belonged to serotype 23F and was autolysin defective, H8 belonged to serotype 13F and had intact autolysin. These strains were not clonally related as determined by pulsed-field gel electrophoresis of chromosomal DNA. In agreement with previous reports, both isolates showed pairing of TIGR4 vex2 with R6 pep27 and had two identical amino acid substitutions, Q441K in vncS and N25D in vex2. These findings indicate that two VTSP strains have emerged independently in Korea, suggesting a prevalence rate of 1.1%. The emergence of VTSP would be a serious threat in Korea, where there are significant rates of penicillin resistance in S. pneumoniae. Monitoring of the prevalence of VTSP and further investigation of the clinical relevance of VTSP are warranted.

Characterization of DNA Damage in Yeast Apoptosis Induced by Hydrogen Peroxide, Acetic Acid, and Hyperosmotic Shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.

Extended Spectrum β-Lactamase (ESBL) in <i>Klebsiella Pneumoniae</i> Isolates from Septicaemic Children in Ibadan, Nigeria

Blood culture was performed on 204 children (aged 1 day – 12 years) who presented at the University College Hospital (UCH) Ibadan, Nigeria, with fever. Eighty-four (41.3%) out of 204 blood cultures were positive. Staphylococcus aureus was the commonest bacterial agent isolated and accounted for (50%) of all the isolates. Klebsiella spp and Salmonella spp each accounted for 21.4% and 10.7% respectively. Other bacterial agents isolated included Escherichia coli, Enterobacter spp. Citrobacter spp. Pseudomonas aeruginosa , Streptococcus Pneumoniae and Serratia spp. Two of the children died in spite of early use of appropriate antibiotics as determined by antibiotic susceptibility testing. Phenotypic and molecualr investigation showed extended-spectrum β-lactamase (ESBL) producing K. pneumoniae to be implicated in the death of the children. Several other K. pneumoniae isolates recovered from blood samples expressed ESBLs and the isolates were shown by pulsed field gel electrophoresis of chromosomal DNA to be genetically unrelated. KEY WORDS: Klebsiella pneumoniae, septicaemic, ESBL. Nigerian Journal of Health and Biomedical Sciences Vol.3(2) 2004: 79-84

Comparison of the Bordetella pertussis and Bordetella parapertussis isolates circulating in Saint Petersburg between 1998 and 2000 with Russian vaccine strains.

We analyzed the Bordetella pertussis and Bordetella parapertussis isolates circulating in Saint Petersburg that were collected between 1998 and 2000 and compared them with isolates collected 40 years ago and Russian vaccine strains. The analysis involved serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion with XbaI and SpeI, and sequencing of the ptxS1 and prn genes, which encode the S1 subunit of the pertussis toxin and the major adhesin pertactin, respectively. The Russian isolates were classified in five of the six pulsed-field gel electrophoresis groups identified in other European countries. The B. pertussis isolates currently circulating in Saint Petersburg differed from the Russian whole-cell vaccine strains and the isolates collected in the prevaccine era. However, their repartition in the major pulsed-field gel electrophoresis groups was slightly different from that of isolates collected in countries that have had a high level of vaccine coverage for a long time, probably because the level of vaccine coverage in Saint Petersburg has increased only recently, after decreasing until the early 1990s. Most of the B. parapertussis isolates studied were similar to those circulating in France. However, some variants were observed, perhaps because B. parapertussis infections are more common in children in this area.

Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure to Bacillus thuringiensis that relates in general to crystal protein gene content

Seventy strains of Bacillus thuringiensis representing 21 serovars were allocated to 38 genomic groups using pulsed field gel electrophoresis (PFGE) of restriction enzyme-digested DNA. There was a broad correlation between PFGE type and serotype for serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, sotto, thuringiensis, and tolworthi, although some serovars included atypical strains. Serovars canadensis and entomocidus were heterogeneous. Detection of crystal protein genes by polymerase chain reaction indicated an approximate correlation between PFGE type and cry gene complement. For example, cry1 products were amplified from DNA from PFGE type 17 strains of serovar aizawai and from PFGE type 23 strains of serovar tolworthi but not from a PFGE 18 strain of aizawai nor from a PFGE type 24 strain of tolworthi. These data suggest a clonal population structure to B. thuringiensis with some consistency of Cry-plasmid composition within PFGE types.

Application of molecular techniques in wine microbiology

Wine fermentation is a complex microbiological process in which yeasts predominate. It is long debated whether yeasts occurring on the surface of grapes or the resident yeasts on the winery equipment play the primary role in conducting the fermentation. The origin, development, changes and succession of various yeast species can be followed using specific molecular techniques allowing the differentiation and typing of yeast strains. Techniques such as pulsed field gel electrophoresis of chromosomal DNA, restriction fragment length polymorphism analysis, and polymerase chain reaction (PCR)-based methods have recently been employed in studying the microbiology of wine making. These shed new light on the dynamics of fermentation started spontaneously or directed by the inoculation of starter cultures.

Polymorphism of Bordetella pertussis isolates circulating for the last 10 years in France, where a single effective whole-cell vaccine has been used for more than 30 years.

We compared Bordetella pertussis isolates collected in France over the last 10 years, the vaccine strains used for more than 30 years, and isolates collected before the introduction of generalized vaccination. The analysis included serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion with XbaI and SpeI, and sequencing of the pt S1 gene, encoding the S1 subunit of pertussis toxin, and the prn gene, encoding the adhesin pertactin. We found that the incidence of infection increases every 3 years. Ninety-five per cent of the isolates analyzed express type 3 fimbriae. Most of the isolates circulating since 1991, unlike the vaccinal strains, express a type A pertussis toxin and a type 2 pertactin. The isolates could be classified into five major groups by pulsed-field gel electrophoresis. Most of these groups correlated with the pertactin type expressed by the isolates. Pulsed-field gel electrophoresis is more discriminative than sequencing particular genes since it could differentiate isolates expressing type 2 pertactin into two subgroups: those circulating in 1993 to 1997 and those circulating in 1997 to 2001. This observation suggests that there has been continuous evolution of the B. pertussis population.

Evidence that hospital hygiene is important in the control of methicillin-resistant Staphylococcus aureus

Observational and microbiological data were collected from the patients and environment of a male general surgical ward over a period of 27 months from January 1998. Isolates of methicillin-resistant Staphylococcus aureus (MRSA) from patients and environment were typed by antibiogram, bacteriophage and pulsed field gel electrophoresis of chromosomal DNA. In September 1999, an intervention was put in place which included increasing the domestic cleaning time by 57 hours per week, with emphasis on removal of dust by vacuum cleaning, and allocation of responsibility for the routine cleaning of shared medical equipment.From January 1998 to September 1999, despite standard infection control measures (emphasis on hand hygiene, isolation of affected patients and staggered closure and cleaning of ward bays), 69 patients acquired a strain of E-MRSA16. This strain was also widespread in the ward environment. Typing confirmed that isolates from patients and environment were indistinguishable from one another and that the outbreak was due to a single strain. This strain was responsible for postoperative infection in approximately one third of the patients who acquired it. In the six months following the intervention, only three patients were colonized with the outbreak MRSA and monthly surveys failed to detect this strain in the environment. Thorough and continuous attention to ward hygiene and removal of dust was needed, to terminate a prolonged outbreak of MRSA infection on a general surgical ward, in addition to standard infection control measures. Control of hospital-acquired infection with MRSA requires a combination of measures, none of which are completely effective in isolation.

Pulsed-field gel electrophoresis of chromosomal DNA of Saccharomyces pastorianus after exposure to x-rays (30-50 keV) and neutrons (14 MeV)

Chromosomes of budding yeast Saccharomyces pastorianus were used to determine the extent of DNA double-strand breaks (DSBs) induced by x-rays (30-50 keV) and 14 MeV neutrons. The yeast chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and the proportion of unbroken molecules corresponding to the largest chromosome no. IV (1500 kbp) was used to calculate the DSB frequency assuming a random distribution of hits. To determine the protective contribution of the cell environment, chromosomes embedded in agarose plugs as well as intact yeast cells, were irradiated under conditions completely inhibiting DNA repair. Following irradiation, the intact cells were also embedded in agarose plugs and the chromosomes isolated to perform PFGE. All radiation experiments resulted in a linear dose-effect curve for DSBs. For both radiation qualities, the yield of DSBs for exposed isolated chromosomes exceeded that for intact yeast cells by a factor of 13. The relative biological effectiveness (RBE) of 14 MeV neutrons in the induction of DNA DSBs was about 2.5. This figure was found to be identical for the in vivo and in vitro exposure of yeast chromosomes (neutrons 36.7 and 2.8, x-rays 14.5 and 1.1 x 10(-8) DSB x Bp-1 Gy-1 for isolated DNA and intact cells, respectively).

Genetic reidentification of the pectinolytic yeast strain SCPP as Saccharomyces bayanus var. uvarum.

Using genetic hybridization analysis, pulsed-field gel electrophoresis of chromosomal DNA and PCR/RFLP analysis of the MET2 gene, we reidentified 11 Champagne yeast strains. Two of them, SCPP and SC4, were found to belong to Saccharomyces bayanus var. uvarum and the remaining strains to S. cerevisiae. Strain

Macrolide resistance mechanisms and expression of phenotypes among Streptococcus pneumoniae circulating in Italy

In Italy, macrolide-resistant pneumococci have been isolated at a rate increasing from 6% in 1993 to 31.7% in 1998. A collection of 161 erythromycin-resistant Streptococcus pneumoniae recovered between 1993 and 1997 has now been phenotypically and genotypically characterized. Approximately 90% of these microorganisms possessed a constitutive MLS(B) mechanism of resistance. PCR detected ermB and mefE genes in strains showing MLS(B) and M phenotypes, respectively. Using pulsed-field gel electrophoresis of chromosomal DNA, one dominant restriction profile and its variations were detected in 51 S. pneumoniae isolates collected from different locations, indicating the circulation of a clone characterized by the possession of a great ability to spread.

Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration.

Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified. A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to-tail tandem array. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype. Telomeric fingerprinting of genomic digests of P. paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus.

Community-acquired Legionnaires' disease following minimal exposure to a contaminated source

A case of community-acquired Legionnaires' disease (LD) is described. The source of infection was traced to a push-on tap at the end of a long spur from the hot circulation system in a large old residential building which had been unoccupied for several weeks. Legionella pneumophila serogroup 1 subgroup Pontiac was isolated from the patient's sputum and from the contaminated water supply. Isolates were shown to be indistinguishable from one another when subtyped by pulsed field gel electrophoresis of chromosomal DNA.

Correlation of six methods for typing nosocomial isolates of Acinetobacter baumannii

A comparative study of biotyping, antimicrobial susceptibility, whole-cell protein analysis, plasmid analysis, pulsed-field gel electrophoresis of chromosomal DNA and polymerase chain reaction with arbitrary primers of Acinetobacter baumannii isolates from three large hospitals was performed to determine the best markers for epidemiological purposes. Ninety-two isolates were included: 38 belonged to a previously described outbreak and 54 were randomly selected from sporadic cases of infection. Biotyping, whole-cell protein and plasmid analysis were the least discriminatory methods, whereas antimicrobial susceptibility and polymerase chain reaction with arbitrary primers showed moderate discriminatory power. Typing based on pulsed-field gel electrophoresis of chromosomal DNA appeared to be the best discriminatory method (discrimination index of 0.9623). The addition of polymerase chain reaction with arbitrary primers or antimicrobial susceptibility to pulsed-field gel electrophoresis of chromosomal DNA did not further increase the discriminatory power.

Chromosomal polymorphism of MEL genes in some populations of Saccharomyces cerevisiae

Using pulsed-field gel electrophoresis of chromosomal DNA and hybridization with the MEL1 probe, we determined the chromosomal locations of polymeric α-galactosidase genes in monosporic cultures of natural strains of Saccharomyces cerevisiae. An unusual phenomenon consisting of an accumulation of the MEL genes has been found in some specific Saccharomyces cerevisiae populations. Many strains possessed a new MEL gene located on chromosome I.

Chromosomal polymorphism of MEL genes in some populations of Saccharomyces cerevisiae.

Using pulsed-field gel electrophoresis of chromosomal DNA and hybridization with the MEL1 probe, we determined the chromosomal locations of polymeric alpha-galactosidase genes in monosporic cultures of natural strains of Saccharomyces cerevisiae. An unusual phenomenon consisting of an accumulation of the MEL genes has been found in some specific Saccharomyces cerevisiae populations. Many strains possessed a new MEL gene located on chromosome I.

Evaluation and characterization of multiresistant Enterococcus faecium from 12 U.S. medical centers.

Forty-two Enterococcus faecium isolates resistant to ampicillin, penicillin, gentamicin, streptomycin, vancomycin, and teicoplanin (VanA phenotype) from 12 U.S. medical centers were analyzed by pulsed-field gel electrophoresis of chromosomal DNA. The isolates were tested for susceptibility to 12 alternative drugs. The results indicated both intrahospital and interhospital diversity among multiresistant vanA enterococcal isolates. Furthermore, the finding of isolates with identical pulsed-field gel electrophoresis patterns in different centers strongly suggests some interhospital clonal transmission.