DNA Pulsed-field Gel Electrophoresis Research Articles

Molecular epidemiology of clinically high-risk Pseudomonas aeruginosa strains: Practical overview.

In recent years, numerous outbreaks of multidrug-resistant Pseudomonas aeruginosa have been reported across the world. Once an outbreak occurs, besides routinely testing isolates for susceptibility to antimicrobials, it is required to check their virulence genotypes and clonality profiles. Replacing pulsed-field gel electrophoresis DNA fingerprinting are faster, easier-to-use, and less expensive polymerase chain reaction (PCR)-based methods for characterizing hospital isolates. P. aeruginosa possesses a mosaic genome structure and a highly conserved core genome displaying low sequence diversity and a highly variable accessory genome that communicates with other Pseudomonas species via horizontal gene transfer. Multiple-locus variable-number tandem-repeat analysis and multilocus sequence typing methods allow for phylogenetic analysis of isolates by PCR amplification of target genes with the support of Internet-based services. The target genes located in the core genome regions usually contain low-frequency mutations, allowing the resulting phylogenetic trees to infer evolutionary processes. The multiplex PCR-based open reading frame typing (POT) method, integron PCR, and exoenzyme genotyping can determine a genotype by PCR amplifying a specific insertion gene in the accessory genome region using a single or a multiple primer set. Thus, analyzing P. aeruginosa isolates for their clonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple-locus targeting methods with other methods that can identify specific virulence and antimicrobial genes. Software packages such as eBURST, R, and Dendroscope, which are powerful tools for phylogenetic analyses, enable researchers and clinicians to visualize clonality associations in clinical isolates.

Evaluating Gulls as Potential Vehicles of Salmonella enterica Serotype Newport (JJPX01.0061) Contamination of Tomatoes Grown on the Eastern Shore of Virginia

Salmonella enterica serovar Newport pattern JJPX01.0061 has been identified as causing several multistate outbreaks in the last 10 years, primarily due to contamination of tomatoes grown in Virginia. The goal of this study was to evaluate gulls as a potential vehicle of S. Newport pattern 61 contamination for tomatoes grown on the Eastern Shore of Virginia. Gull fecal samples were collected at four sites in eastern Virginia for 3 months (May to July) in 2012, resulting in 360 samples, among which Salmonella was isolated from 62 samples. Twenty-two serotypes and 26 pulsed-field gel electrophoresis DNA fingerprint patterns, including S. Newport pattern 61, were identified. All of the patterns that were isolated multiple times, with the exception of S. Newport patterns JJPX01.0030 and JJPX01.0061, were clustered in time and geographical location. These results strongly suggest that both patterns of S. Newport are endemic to sites on the Eastern Shore where gulls were sampled. This study provides additional information regarding the epidemiology of S. Newport pattern 61 in Virginia and how tomatoes sold interstate may become contaminated in the field.

Escherichia coli contamination and health aspects of soil and tomatoes (Solanum lycopersicum L.) subsurface drip irrigated with on-site treated domestic wastewater

Faecal contamination of soil and tomatoes irrigated by sprinkler as well as surface and subsurface drip irrigation with treated domestic wastewater were compared in 2007 and 2008 at experimental sites in Crete and Italy. Wastewater was treated by Membrane Bio Reactor (MBR) technology, gravel filtration or UV-treatment before used for irrigation. Irrigation water, soil and tomato samples were collected during two cropping seasons and enumerated for the faecal indicator bacterium Escherichia coli and helminth eggs. The study found elevated levels of E. coli in irrigation water (mean: Italy 1753 cell forming unit (cfu) per 100 ml and Crete 488 cfu per 100 ml) and low concentrations of E. coli in soil (mean: Italy 95 cfu g−1 and Crete 33 cfu g−1). Only two out of 84 tomato samples in Crete contained E. coli (mean: 2700 cfu g−1) while tomatoes from Italy were free of E. coli. No helminth eggs were found in the irrigation water or on the tomatoes from Crete. Two tomato samples out of 36 from Italy were contaminated by helminth eggs (mean: 0.18 eggs g−1) and had been irrigated with treated wastewater and tap water, respectively. Pulsed Field Gel Electrophoresis DNA fingerprints of E. coli collected during 2008 showed no identical pattern between water and soil isolates which indicates contribution from other environmental sources with E. coli, e.g. wildlife. A quantitative microbial risk assessment (QMRA) model with Monte Carlo simulations adopted by the World Health Organization (WHO) found the use of tap water and treated wastewater to be associated with risks that exceed permissible limits as proposed by the WHO (1.0 × 10−3 disease risk per person per year) for the accidental ingestion of irrigated soil by farmers (Crete: 0.67 pppy and Italy: 1.0 pppy). The QMRA found that the consumption of tomatoes in Italy was deemed to be safe while permissible limits were exceeded in Crete (1.0 pppy). Overall the quality of tomatoes was safe for human consumption since the disease risk found on Crete was based on only two contaminated tomato samples. It is a fundamental limitation of the WHO QMRA model that it is not based on actual pathogen numbers, but rather on numbers of E. coli converted to estimated pathogen numbers, since it is widely accepted that there is poor correlation between E. coli and viral and parasite pathogens. Our findings also stress the importance of the external environment, typically wildlife, as sources of faecal contamination.

Methicillin-resistant Staphylococcus aureus DNA electrophoretic pattern: temporal changes in an endemic hospital environment.

To describe the analysis of geographical and temporal distribution of DNA profiles determined by pulsed-field gel electrophoresis (PFGE) of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from hospitalized patients in a tertiary care university hospital in Brazil. Ninetynine samples of MRSA obtained from 89 patients in the period 1999-2004 were studied. MRSA strains were isolated from central venous catheters (33 isolates) and bloodstream infections (66 strains). PFGE with 20 units of SmaI restriction endonuclease was used for genomic typing. Analysis of DNA PFGE of 99 strains of MRSA revealed 26 profiles and their respective related profiles. The mean time interval for detecting MRSA infection was 26 days from hospital admission. Forty-nine patients (57.6%) had a recent hospitalization. The DNA PFGE MRSA profiles were distributed in three clonal groups-I, II, and III-according to the period of time when the MRSA strains were isolated. DNA PFGE MRSA profiles were spread homogeneously through all hospital wards. Changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospital ward or any specific area of the hospital.

Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks

Purpose The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. Materials and methods We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. Results We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. Conclusions These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

Characterization of the Genomes of a Diverse Collection of Salmonella enterica Serovar Typhimurium Definitive Phage Type 104

Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) has caused significant morbidity and mortality in humans and animals for almost three decades. We completed the full DNA sequence of one DT104 strain, NCTC13348, and showed that significant differences between the genome of this isolate and the genome of the previously sequenced strain Salmonella serovar Typhimurium LT2 are due to integrated prophage elements and Salmonella genomic island 1 encoding antibiotic resistance genes. Thirteen isolates of Salmonella serovar Typhimurium DT104 with different pulsed-field gel electrophoresis (PFGE) profiles were analyzed by using multilocus sequence typing (MLST), plasmid profiling, hybridization to a pan-Salmonella DNA microarray, and prophage-based multiplex PCR. All the isolates belonged to a single MLST type, sequence type ST19. Microarray data demonstrated that the gene contents of the 13 DT104 isolates were remarkably conserved. The PFGE DNA fragment size differences in these isolates could be explained to a great extent by differences in the prophage and plasmid contents. Thus, here the nature of variation in different Salmonella serovar Typhimurium DT104 isolates is further defined at the gene and whole-genome levels, illustrating how this phage type evolves over time.

Molecular Epidemiology of Staphylococcus saprophyticus Isolated from Women with Uncomplicated Community-Acquired Urinary Tract Infection

Staphylococcus saprophyticus is a common cause of urinary tract infections (UTIs) in women. Little is known about the molecular epidemiology of S. saprophyticus UTIs. In the current study, we compared 76 isolates of S. saprophyticus prospectively isolated from women with uncomplicated UTI participating in a randomized placebo-controlled treatment trial performed in northern Sweden from 1995 to 1997 with 50 strains obtained in 2006 from five different locations in northern Europe with pulsed-field gel electrophoresis (PFGE). The aim was to elucidate the molecular epidemiology of this uropathogenic species and to investigate whether specific clones are associated with UTI in women. A total of 47 different PFGE profiles were detected among the 126 analyzed isolates. Ten clusters consisting of 5 to 12 isolates each showing PFGE DNA similarity of >85% were identified. Several clusters of genetically highly related isolates were detected in the original trial as well as among isolates obtained during 2006 from different locations. In the original trial, clonal persistence was found among 16 of 21 (76%) patients examined in the placebo group at follow-up 8 to 10 days after inclusion, indicating a low spontaneous short-time bacteriological cure rate. We conclude that multiple clones of S. saprophyticus were causing lower UTIs in women. The result suggests that some human-pathogenic clones of S. saprophyticus are spread over large geographical distances and that such clones may persist over long periods of time.

Recovery of Salmonella, Listeria monocytogenes, and Mycobacterium bovis from Cheese Entering the United States through a Noncommercial Land Port of Entry

A joint multiagency project was initiated in response to a Salmonella outbreak in San Diego County, California, in 2004. Samples of cheese were collected during four 1-day operations at the San Ysidro port of entry, along the United States–Mexico border. Surveyed participants were persons crossing the border as pedestrians or in vehicles who had a minimum of 2.27 kg of cheese, which may suggest a potential diversion to illegal marketing. In addition, data were collected about the cheese to identify risk factors for cheese contamination. Two hundred four cheese samples were submitted to the California Animal Health and Food Safety Laboratory System–San Bernardino Branch and analyzed for potential food pathogens. Ninety-four percent (190 of 203) of the samples tested positive for alkaline phosphatase. Salmonella was detected from 13% (27 of 204) of the samples comprising 11 serogroups and 28 serotypes. Pulsed-field gel electrophoresis DNA fingerprinting analysis, performed following standardized methods, determined that an isolate obtained from this study had an indistinguishable pattern from a recent Salmonella enterica serovar Typhimurium var. Copenhagen epidemic in the San Diego County that was linked to 14 illnesses. Listeria spp. were detected from 4% (8 of 204) of the samples, and of these, half were identified as L. monocytogenes. Escherichia coli O157:H7 was not detected from any of the samples. Mycobacterium bovis was detected from one panela-style cheese sample. Nine additional samples yielded Mycobacterium spp.

Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast

Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast

Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of DNA from Mammalian Cells and Tissues

Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of DNA from Mammalian Cells and Tissues

The effects of combined gentamicin–hydroxyapatite coating for cementless joint prostheses on the reduction of infection rates in a rabbit infection prophylaxis model

Infections remain a critical issue in total joint arthroplasty. Addition of antibiotics to bone cement was shown to significantly improve antimicrobial prophylaxis in cemented joint arthroplasty. In cementless joint arthroplasty a comparable prophylaxis by local antibiotics has not been possible yet. The aim of the current study was to investigate the antimicrobial effect of two different gentamicin-hydroxyapatite (HA) coatings for cementless prostheses in a rabbit infection model. Staphylococcus aureus with a dose of 10(7) CFUs was inoculated into the intramedullary canal of the tibia of 30 rabbits followed by the implantation of standard steel HA K-wires (n=10), steel K-wires coated with a gentamicin-HA combination (n=10), and steel K-wires coated with a gentamicin-RGD-HA combination (n=10), respectively. The animals were sacrificed after 28 days and clinical, histological and microbiological assessment on the bone and on the removed K-wire itself by agar plating and DNA-pulsed field gel electrophoresis were carried out to detect infection. There was a statistically significant reduction of infection rates by both gentamicin-coating types (0 infections in both groups) compared to standard HA coating (7 infections in 8 animals; 2 animals were lost due to acute diarrhea) (p<0.001). An excellent correlation between agar plating testing results of the K-wires and of the bone samples was found. Detailed histology showed cortical lysis, abscess and sequester formation in the infected animals. Both gentamicin-coating types showed significant improvement of infection prophylaxis compared to standard HA coating and, therefore, this coating technology could help to improve infection prophylaxis in cementless total joint arthroplasty. In further studies biocompatibility of the coatings has to be assessed.

Genotyping of Ureaplasma diversum isolates using pulsed-field electrophoresis

Ureaplasma diversum has been associated with reproductive disorders in cattle and in the present study genotypic variations among U. diversum isolates obtained from the vaginal mucus of healthy cattle and sick animals were analyzed by enzymatic digestion and pulsed-field gel electrophoresis (PFGE). The influence of time and broth volume was important in obtaining sufficient cell sediment and DNA for PFGE. The method presented a high discriminatory power and satisfactory reproducibility for the analysis of detected variations among U. diversum isolates and strains. Different band profiles and wide genotypic heterogeneity were detected but no association between DNA polymorphism and sick or healthy animals could be established.

First general outbreak of Verocytotoxin-producing Escherichia coli O157 in Denmark.

This report describes the first general outbreak of verocytotoxin-producing E. coli (VTEC) in Denmark. Twenty five patients, 18 children and seven adults, with culture-confirmed VTEC O157:H- infection and indistinguishable pulsed-field gel electrophoresis DNA profiles, were identified during a six month period from September 2003 to March 2004. The outbreak strain possessed the virulence genes: eae, vtx1 and vtx2c. All patients but one presented with diarrhoea; none developed haemolytic uraemic syndrome. The outbreak was restricted to Copenhagen and surrounding areas. A case-control study including 11 cases and 55 matched controls revealed an association between VTEC O157:H- infection and shopping in a specific supermarket chain in Copenhagen and surrounding area, matched odds ratio (OR): 8.7 (95% confidence interval (CI): 1.1-71). After exclusion of three assumed secondary cases, only consumption of a particular kind of organic milk from a small dairy was associated with disease OR: 8.7 (95% CI 1.6-48). Environmental and microbiological investigations at the suspected dairy did not confirm the presence of the outbreak strain, but the outbreak stopped once the dairy was closed and thoroughly cleaned.

A diversity of bacteriophage forms and genomes can be isolated from the surface sands of the Sahara Desert

The surface sands of the Sahara Desert are exposed to extremes of ultraviolet light irradiation, desiccation and temperature variation. Nonetheless, the presence of bacteria has recently been demonstrated in this environment by cultivation methods and by 16S rDNA analyses from total DNA isolated from surface sands. To discern the presence of bacteriophages in this harsh environment, we searched for extracellular phages and intracellularly located phages present as prophages or within pseudolysogens. Mild sonication of the sand, in different liquid culture media, incubated with and without Mitomycin-C, was followed by differential centrifugation to enrich for dsDNA phages. The resulting preparations, examined by electron microscopy, revealed the presence of virus-like particles with a diversity of morphotypes representative of all three major double-stranded DNA bacteriophage families (Myoviridae, Siphoviridae and Podoviridae). Moreover, pulsed-field gel electrophoresis of DNA, extracted from the enriched bacteriophage preparations, revealed the presence of distinct bands suggesting the presence of putative dsDNA phage genomes ranging in size from 45 kb to 270 kb. Characterization of the bacteriophages present in the surface sands of the Sahara Desert extends the range of environments from which bacteriophages can be isolated, and provides an important point of departure for the study of phages in extreme terrestrial environments.

Proteinase PI and lactococcin A genes are located on the largest plasmid inLactococcus lactissubsp.lactisbv. diacetylactis S50

Lactococcus lactis subsp. lactis bv. diacetylactis S50 produces a lactococcin A-like bacteriocin named bacteriocin S50, and cell envelope-associated PI-type proteinase activity. This strain harbours 3 small size plasmids: pS6 (6.3 kb), pS7a (7.31 kb), and pS7b (7.27 kb). Plasmid curing using a combination of novobiocin treatment (10 microg.mL-1) and sublethal temperature (40 degrees C) resulted in a very low yield (0.17%) of Prt-, Bac-, Bacs derivatives, which retained all 3 small size resident plasmids. Pulsed-field gel electrophoresis of DNA isolated from the strain S50 and cured derivatives in combination with restriction enzyme analysis and DNA-DNA hybridization revealed that S50 contains 2 additional large plasmids: pS140 (140 kb) and pS80 (80 kb). Conjugation experiments using strain S50 as a donor and various lactococcal recipients resulted in Prt+, Bac+, Bacr transconjugants. Analysis of these transconjugants strongly indicated that plasmid pS140 harbours the prt and bac genes encoding proteinase and bacteriocin production, and immunity to bacteriocin, since each Prt+, Bac+, Bacr tranconjugant contained pS140. Accordingly, none of the Prt-,Bac-, Bacs transconjugants contained this plasmid. pS140 was a self-transmissible conjugative plasmid regardless of the host lactococcal recipient used in the test. Frequency of conjugation of plasmid pS140 did not depend on either the donor or recipient strain.

Surveillance of severe invasive group-G streptococcal infections and molecular typing of the isolates in Japan.

The number of patients with severe invasive group-G streptococcal (Streptococcus dysgalactiae subsp. equisimilis) infections has been increasing in Japan. The emm genotypes and SmaI-digested pulsed-field gel electrophoresis DNA profiles were variable among the strains isolated, suggesting there has not been clonal expansion of a specific subpopulation of strains. However, all strains carried scpA, ska, slo and sag genes, some of which may be involved in the pathogenesis of the disease.

Chromosome DNA fragmentation and excretion caused by defective prophage gene expression in the early-exponential-phase culture ofBacillus subtilis

Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria-Bertani medium. Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands. Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis. Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome. SDS-PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA. Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome. Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion. Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168. The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.

Determining the One, Two, Three, or Four Long and Short Loci of Human Complement C4 in a Major Histocompatibility Complex Haplotype Encoding C4A or C4B Proteins

The complex genetics of human complement C4 with unusually frequent variations in the size and number of C4A and C4B, as well as their neighboring genes, in the major histocompatibility complex has been a hurdle for accurate epidemiological studies of diseases associated with C4. A comprehensive series of novel or improved techniques has been developed to determine the total gene number of C4 and the relative dosages of C4A and C4B in a diploid genome. These techniques include (1) definitive genomic restriction-fragment-length polymorphisms (RFLPs) based on the discrete duplication patterns of the RCCX (RP-C4-CYP21-TNX) modules and on the specific nucleotide changes for C4A and C4B isotypes; (2) module-specific PCR to give information on the total number of C4 genes by comparing the relative quantities of RP1- or TNXB-specific fragments with TNXA-RP2 fragments; (3) labeled-primer single-cycle DNA polymerization procedure of amplified C4d genomic DNA for diagnostic RFLP analysis of C4A and C4B; and (4) a highly reproducible long-range-mapping method that employs PmeI-digested genomic DNA for pulsed-field gel electrophoresis, to yield precise information on the number of long and short C4 genes in a haplotype. Applications of these vigorously tested techniques may clarify the roles that human C4A and C4B gene-dosage variations play in infectious and autoimmune diseases.

Pulsed-Field Gel Elec rophoresis Pa ern Recogni ion of Bac erial DNA: A Systemic Approach

Pulsed-Field Gel Electrophoresis (PFGE) images contain ‘banding pattern’ information that can be used in bacterial identification.A systemic approach was designed to target the bacterial DNA PFGE pattern recognition problem that requires image processing, feature extraction, data pre-processing, classification and final decision-making. This paper describes the research, development and testing of a complete system to address this identification problem, including Receiver Operating Characteristic Curve (ROC) analysis. Variations due to gel preparation in various laboratories by different technicians are considered. The approach was applied to recognition of E. coli O157:H7 bacteria with excellent results.

Dynamics of Salmonella Contamination in a Commercial Quail Operation

Control of carcass contamination requires knowledge of the source and dynamics of spread of Salmonella in commercial poultry production. We examined Salmonella contamination at a U.S. commercial quail operation. Pulsed-field gel electrophoresis (PFGE) was used to type isolates in order to trace Salmonella throughout this production environment. During a 6-mo survey, Salmonella serotypes hadar, typhimurium, typhimurium variant Copenhagen, and paratyphi were encountered within this poultry operation. Ninety-four percent of the Salmonella isolated from breeder and production houses and from carcass rinses belonged to Salmonella serotypes typhimurium variant Copenhagen and hadar. There were six distinct S. typhimurium variant Copenhagen genetic types, as identified by PFGE, present within this particular poultry operation. Seventy-nine percent of S. typhimurium variant Copenhagen identified from the environment of the breeder and production houses produced the same PFGE pattern. Thirty-eight percent of S. typhimurium Copenhagen isolated from carcass rinses and the breeder house shared the same PFGE DNA pattern. This study demonstrates the transmission of salmonellae throughout this production environment, from the breeders to their progeny and to the birds ultimately processed for human consumption.