Pulsed-field Electrophoresis Research Articles

Enfermedades invasoras por Streptococcus pyogenes 2005-2013: Hospital Pediátrico del Centro Hospitalario Pereira Rossell, Uruguay

Streptococcus pyogenes infection causes a wide spectrum of clinical manifestations. Invasive disease (ID) is defined by the isolation of the microorganism from sterile sites. To analyze the clinical, epidemiological and molecular characteristics of ID by S.pyogenes in children hospitalized at Pediatric Hospital Pereira Rossell, from January 2005 to January 2013. A descriptive retrospective study was done in cases with isolation of S.pyogenes from sterile sites. Epidemiological variables, disease characteristics, laboratory parameters, treatment regimen received, hospitalization days and clinical outcome were analyzed. The presence of 4 genes encoding for virulence factors and chromosome profile studied by pulsed-field electrophoresis were done in the isolated strains. A total of 42 cases (rate: 4.6 out of 10,000 admissions) were detected, from which 32 were analyzed. Average age was 44.7 months (14/32 < 2 years of age). In 5 cases, the portal of entry was identified. Clinical presentations were: osteoarticular infections (n = 15), shock (n = 6), skin and soft-tissue infections (n = 5), pneumonia (n = 3) and bacteremias (n = 3). Twenty cases required surgical procedures and 13 required intensive care admission. Average hospital stay was 17 days and one patient died. Molecular studies were performed in five strains; 4 different toxin profiles and pulsotypes were identified. The incidence of ID at our hospital is similar to other series in the region. A better knowledge of clinical presentation and its relation with molecular characteristics represents a challenge.

Finding of dairy yeasts Kluyveromyces lactis var. lactis in natural habitats

Well-known yeasts Kluyveromyces lactis var. lactis, which are usually associated with dairy prod- ucts, were discovered in nature (in woodland park soil under Impatiens glandulifera Royle plants). Reliable identification of the yeasts was carried out using physiological criteria (lactose and maltose utilization) and molecular markers (nucleotide sequence of the 5.8S-ITS rDNA fragment, pulsed-field electrophoresis, and Southern hybridization of chromosomal DNA with the LAC4 probe). Ecology of KI. lactis var. lactis is discussed.

Driving forces of vancomycin-resistant E. faecium and E. faecalis blood-stream infections in children.

BackgroundRates of invasive vancomycin-resistant Enterococcus (VRE) in the USA remains on the rise. Efforts to control vancomycin use and nosocomial transmission have had limited success in halting the spread of this pathogen. The role of antibiotic exposure remains a topic of controversy. We evaluated the association between emergence of VRE-blood-stream infections (BSI), aggregate and individual-patient vancomycin- exposure, and clonal transmission of VRE at an academic pediatric tertiary care hospital.MethodsE. faecium and E. faecalis isolates recovered from blood specimens from hospitalized children from 2003–2010 were retrieved from the microbiology database. Aggregate vancomycin use and individual-patient vancomycin exposure 6 months preceding each event of bacteremia were recorded. Pulse-field electrophoresis was performed on selected VRE isolates.ResultsOf 151 episodes of E. faecium and E. faecalis BSI among hospitalized children <18 years of age, 9% (14) were due to VRE. Of these, 5 (36%) were due to nosocomial transmission. Aggregate (r .19, P = 0.3) and individual-patient vancomycin-exposure (X 2 = .26; P = .87) were not associated with VRE-BSI. On bivariate analysis, OR for developing VRE-BSI among patients infected with clonal isolates was 36 (P < .0001). Infection control interventions, rather than antimicrobial stewardship interventions to decrease vancomycin use, proved to be effective in reducing the rates of VRE-BSI.ConclusionsIn our experience, VRE-BSI was associated with nosocomial transmission and was independent of aggregate and individual-patient vancomycin-exposure. Molecular epidemiology is a crucial tool to differentiate the role of nosocomial transmission and antibiotic exposure in the emergence of invasive VRE infections among hospitalized children.

Epidemic Clostridium difficile strains demonstrate increased competitive fitness compared to nonepidemic isolates.

Clostridium difficile infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of C. difficile infection have paralleled the emergence of a specific phylogenetic clade of C. difficile strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput in vitro model of C. difficile infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of C. difficile infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota.

Reprint of “Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray)”

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

Mechanism of DNA trapping in nanoporous structures during asymmetric pulsed-field electrophoresis.

We investigate the trapping mechanism of individual DNA molecules in ordered nanoporous structures generated by crystalline particle arrays. Two requisites for trapping are revealed by the dynamics of single trapped DNA, fully-stretched U/J shapes and hernia formation. The experimental results show there is a stronger possibility for hernias to lead the reorientation upon switching directions of the voltage at high field strengths, where trapping occurs. Fully stretched DNA has longer unhooking times than expected by a classic rope-on-pulley model. We propose a dielectrophoretic (DEP) force reduces the mobility of segments at the apex of the U or J, where field gradients are highest, based on simulations and observations of the trapping force dependence on field strength. A modified model for unhooking time is obtained after the DEP force is introduced. The new model explains the unhooking time data by predicting an infinite trapping time when the ratio of arm length differences (of the U or J) to molecule length Δx/L < β, where β is a DEP parameter that is found to strongly increase with electric field. The DNA polarizability calculated with the DEP model and experimental value of β is of the same magnitude of reported value. The results indicate the tension at the apex of U/J shape DNA is the primary reason for DNA trapping during pulsed field separation, instead of hernias.

Analysis of the genome of a Korean isolate of the Pieris rapae granulovirus enabled by its separation from total host genomic DNA by pulse-field electrophoresis.

BackgroundMost traditional genome sequencing projects involving viruses include the culture and purification of the virus particles. However, purification of virions may yield insufficient material for traditional sequencing. The electrophoretic method described here provides a strategy whereby the genomic DNA of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) could be recovered in sufficient amounts for sequencing by purifying it directly from total host DNA by pulse-field gel electrophoresis (PFGE).Methodology/Principal FindingsThe total genomic DNA of infected P. rapae was embedded in agarose plugs, treated with restriction nuclease and methylase, and then PFGE was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the purified viral DNA. The double-stranded circular genome of PiraGV-K was found to encode 120 open reading frames (ORFs), which covered 92% of the sequence. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (∼99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF 11), involved in the liquefaction of the host, were found in the genome.Conclusions/SignificanceThe recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the electrophoretic method. The method appears to be generally applicable to the analysis of genomes of large viruses.

Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray)

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

Probable nosocomial transmission of listeriosis in neonates

Listeria monocytogenes was isolated in two neonates born consecutively in the same hospital in France. The isolates had indistinguishable pulsed-field electrophoresis profiles. Retrospective epidemiological investigations found no evidence of a food-borne or environmental source. Infection control protocols and decontamination processes were in accordance with standard recommendations. The timing of onset of these infections within the same maternity unit, and the similarity of pulsed-field gel electrophoresis profiles suggests cross-infection of L.monocytogenes between the two neonates.

Aerosolization of Acinetobacter baumannii in a Trauma ICU*

To establish the presence of air contamination with Acinetobacter baumannii in the trauma ICU. Point prevalence microbiological surveillances. A 1,500-bed public teaching hospital in the Miami metro area. Trauma ICU patients. Pulsed field electrophoresis was performed on environmental and clinical isolates to determine the association of any isolates from the air with clinical isolates. Out of 53 patient areas cultured, 12 (22.6%) had their air positive for A. baumannii. The presence of an A. baumannii-positive patient (underneath the plate) was associated with positive air cultures for A. baumannii (11 of 21 [52.4%] vs 0 of 25 [0%]; p < 0.0001). However, we were not able to find differences in air contamination based on the presence of A. baumannii in respiratory secretions versus absence (p = 1.0). Air and clinical isolates were found to be clonally related. Aerosolization of A. baumannii in the ICUs is a concern, and its role in the transmission of this organism among patients should be further clarified.

Nonmonotonous variation of DNA angular separation during asymmetric pulsed field electrophoresis

Asymmetric pulsed field electrophoresis within crystalline arrays is used to generate angular separation of DNA molecules. Four regimes of the frequency response are observed, a low frequency rise in angular separation, a plateau, a subsequent decline, and a second plateau at higher frequencies. It is shown that the frequency response for different sized DNA is governed by the relation between pulse time and the reorientation time of DNA molecules. The decline in angular separation at higher frequencies has not previously been analyzed. Real-time videos of single DNA molecules migrating under high frequency-pulsed electric field show the molecules no longer follow the head to tail switching, ratchet mechanism seen at lower frequencies. Once the pulse period is shorter than the reorientation time, the migration mechanism changes significantly. The molecule reptates along the average direction of the two electric fields, which reduces the angular separation. A freely jointed chain model of DNA is developed where the porous structure is represented with a hexagonal array of obstacles. The model qualitatively predicts the variation of DNA angular separation with respect to frequency.

Large‐scale structural variations linked to the NOTCH4 locus of the Human Major Histocompatibility Complex

The human major histocompability complex (MHC) encodes antigen‐presenting proteins and other proteins critical to the function of the the immune system. It is also the most polymorphic component of the human genome. Variations within the MHC region have been linked to both susceptibility and resistance to multiple autoimmune diseases. The objective of the present study is to determine whether the portion of the MHC linked to the NOTCH4 gene is characterized by large‐scale structural variation and if it is, whether that variation is linked to auto‐immune disease. The NCBI human genome reference sequence indicates that NOTCH4 is contained within a 450 kilobase PmeI restriction fragment. To date, this study has used polymerase chain reaction to isolate NOTCH4 specific probes from cDNA for NOTCH4. The probes will be hybridized with Southern blots of pulsed field electrophoresis gels of PmeI digested genomic DNA isolated from patients with systemic lupus erythematosus and normal controls. This will allow the observation of size variation and imply the possibility of copy number variations, insertions and/or deletion within the region and their possible role in autoimmune disease.

Beta-lactamase production by Kingella kingae in Israel is clonal and common in carriage organisms but rare among invasive strains

The purpose of this study was to investigate the prevalence of β-lactamase and the genomic clonality of a large collection of Kingella kingae isolates from Israeli patients with a variety of invasive infections and asymptomatic pharyngeal carriers. β-lactamase production was studied by the nitrocefin method and the minimum inhibitory concentrations (MICs) of penicillin and amoxicillin-clavulanate were determined by the epsilon (Etest) method. The genotypic clonality of isolates was investigated by pulsed-field electrophoresis (PFGE). β-lactamase was found in 2 of 190 (1.1%) invasive isolates and in 66 of 429 (15.4%) randomly chosen carriage organisms (p < 0.001). Overall, 73 distinct PFGE clones were identified (33 among invasive organisms and 56 among carriage isolates). β-lactamase production was found to be limited to four distinct PFGE clones, which were common among carriage strains but rare among invasive strains, and all organisms in the collection belonging to these four clones expressed β-lactamase. The penicillin MIC of β-lactamase-producing isolates ranged between 0.094 and 2mcg/mL (MIC50: 0.25mcg/mL; MIC90: 1.5mcg/mL) and that of amoxicillin-clavulanate between 0.064 and 0.47mcg/mL (MIC50: 0.125mcg/mL; MIC90: 0.125mcg/mL). The penicillin MIC of β-lactamase non-producing isolates ranged between <0.002 and 0.064mcg/mL (MIC50: 0.023mcg/mL; MIC90: 0.047mcg/mL). Although β-lactamase production is prevalent among K. kingae organisms carried by healthy carriers, the low invasive potential of most colonizing clones results in infrequent detection of the enzyme in isolates from patients with clinical infections. The exceptional presence of β-lactamase among invasive organisms correlates with the favorable response of K. kingae infections to the administration of β-lactamase-susceptible antibiotics.

Reidentification of chromosomal CUP1 translocations in the wine yeasts Saccharomyces cerevisiae

Reciprocal translocations between chromosomes XVI and VIII were revealed in eight Saccharomyces cerevisiae strains (mostly wine ones) using pulse-field electrophoresis of native chromosomal DNAs and their hybridizations with the CUP1 and GAL4 probes. New and reciprocal translocations of at least the gene CUP1 occur at the expense of crossing-over in the hybrids of such strains with the genetic lines of normal karyotype during meiosis. Relationship between these reciprocal translocations and the sulfite (Na2SO3) resistance gene SSU1-R is discussed.

Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ∼170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.

Relationship between frequency and deflection angle in the DNA prism.

The DNA prism is a modification of the standard pulsed-field electrophoresis protocol to provide a continuous separation, where the DNA are deflected at an angle that depends on their molecular weight. The standard switchback model for the DNA prism predicts a monotonic increase in the deflection angle as a function of the frequency for switching the field until a plateau regime is reached. However, experiments indicate that the deflection angle achieves a maximum value before decaying to a size-independent value at high frequencies. Using Brownian dynamics simulations, we show that the maximum in the deflection angle is related to the reorientation time for the DNA and the decay in deflection angle at high frequencies is due to inadequate stretching. The generic features of the dependence of the deflection angle on molecular weight, switching frequency, and electric field strength explain a number of experimental phenomena.

Comparative molecular and antibody typing during the investigation of an outbreak of Legionnaires’ disease

An outbreak of Legionnaires' disease with 113 confirmed cases was reported in the town of Mataró, Spain, in August 2002. In this study, we compared three different typing methods and characterized the clinical isolates by comparing them with other clinical isolates with the same ST from our own database to further characterize the outbreak. In the outbreak, a total of 16 clinical (nine patients) and 32 environmental (from four environmental sources) Legionella pneumophila isolates were analyzed by pulsed-field electrophoresis (PFGE), sequence-based typing (SBT), and monoclonal antibody typing (MAb). We compared the MAb and SBT profiles of the outbreak clinical isolates and other unrelated clinical isolates showing the same ST profile. We obtained seven different PFGE and SBT profiles and six MAb patterns from the outbreak isolates. PFGE and SBT showed 100% concordance during the outbreak. SBT proved to be highly discriminatory, particularly with the addition of the new neuA gene. One PFGE, SBT (ST-37), and Philadelphia profile was observed among the clinical isolates. Using PFGE, this ST37 Philadelphia profile was closely related to other unrelated clinical isolates. These findings suggest that the ST37 Philadelphia profile could be a virulence marker in our area. The combination of the three methodologies was useful to further characterize and obtain additional information on a very explosive outbreak. Despite the minor discrimination of PFGE versus SBT, the two genetic methods are recommended in outbreak investigations. Further studies are currently underway in this area to obtain more definitive conclusions.

Epidemic of Salmonellosis in Passerine Birds in Switzerland With Spillover to Domestic Cats

A die-off of passerine birds, mostly Eurasian siskins (Carduelis spinus), occurred in multiple areas of Switzerland between February and March 2010. Several of the dead birds were submitted for full necropsy. Bacteriological examination was carried out on multiple tissues of each bird. At gross examination, common findings were light-tan nodules, 1 to 4 mm in diameter, scattered through the esophagus/crop. Histologically, a necroulcerative transmural esophagitis/ingluvitis was observed. Bacterial cultures yielded Salmonella enterica subsp. enterica serovar Typhimurium. At the same time, 2 pet clinics reported an unusual increase of domestic cats presented with fever, anorexia, occasionally dolent abdomen, and history of presumed consumption of passerine birds. Analysis of rectal swabs revealed the presence of S. Typhimurium in all tested cats. PFGE (pulsed field electrophoresis) analysis was performed to characterize and compare the bacterial isolates, and it revealed an indistinguishable pattern between all the avian and all but 1 of the feline isolates. Cloacal swabs collected from clinically healthy migrating Eurasian siskins (during autumn 2010) did not yield S. Typhimurium. The histological and bacteriological findings were consistent with a systemic infection caused by S. Typhimurium. Isolation of the same serovar from the dead birds and ill cats, along with the overlapping results of the PFGE analysis for all the animal species, confirmed a spillover from birds to cats through predation. The sudden increase of the number of siskins over the Swiss territory and their persistency during the whole winter of 2009-2010 is considered the most likely predisposing factor for the onset of the epidemic.

Molecular characterization of a new lytic bacteriophage isolated from cheese whey

In this study, we isolated and characterized a lytic Lactococcus lactis bacteriophage from the sera of a failed fermentation. The phage was isolated and cultured in L. lactis subsp. cremoris in M17 medium. The isolated bacteriophage was characterized by multiplex PCR, pulsed-field electrophoresis, DNA restriction digestion, analysis of the N-terminal sequence of the phage major structural protein, transmission electron microscopy and sequencing and analysis of a conserved fragment of its genome. Analysis of the viral genome indicates that its genome is composed of a DNA strand of approximately 48 kb in length, and PCR and microscopy confirmed that IL-P1 belongs to the group of 936-type phages in the family Siphoviridae, which is the most abundant type of lactococcal virus in dairy products worldwide. To our knowledge, this is the first report of a virus within this family that has a presumptive genome larger than 40 kb.

Development and characterization of an isogenic cell line with a radioresistant phenotype

Radiation resistance is a major cause of death in cancer patients. Cancer cells react during radiotherapy by re-programming specific cell functions that may confer resistance to radiation. The understanding of this complex process is hindered due to the lack of appropriate study models. We describe an experimental development of a radioresistant isogenic cancer cell line, and its molecular characterization. A431-cultured cells were irradiated for 7month until 85Gy. Then, a selected single cell was left to grow as stable A431-R cell line. Clonogenic assay was used to determine cell survival, the α and β parameters of the LQ model, and the mean inactivation dose. The DNA repair ability of cells was evaluated by pulsed-field electrophoresis method. Differential effect of fractionated radiation was ultimately tested in xenografts. Furthermore, we used a wound healing assay, Western blot for EGFR, AKT and ERK1/2 and ELISA test for vascular endothelial growth factor (VEGF) secretion. Finally we explored CD44 marker and cell cycle distribution. The established A431-R cell line showed radiation resistance in clonogenic assays, repair of radiation-induced DNA fragmentation and xenografted tumours. The radiation resistance was associated with in vitro higher cell growth and migration, increased levels of former oncoproteins, and secretion of VEGF. In this model, the emergence of radiation resistance was associated with the acquisition of biological traits that support more aggressive behaviour of cancer cells. We have generated a model that will be useful for mechanistic studies and development of rational treatments against radiation resistance in cancer.