Background Production of extended-spectrum β-lactamase (ESBL) is one of the antibiotic resistance strategies in the bacteria. Extended spectrum β-lactamase producing Escherichia coli (E. coli) infections alarmingly increased in recent years in Turkey.Objectives The current study aimed at determining antibiotic resistance and genotypic profiles of ESBL-positive E. coli isolates.Methods Forty-five ESBL-positive E. coli species were isolated from a variety of units both at the Mevlana University Foundation Hospital and the Mevlana University Medical Center at Konya province in Turkey from December 2013 to December 2014. Antibiotic resistance profile was determined by the Kirby-Bauer disk diffusion method. Genotypic profile was determined by pulsed-field gel electrophoresis (PFGE).Results The rate of ESBL production in E. coli strains was 13.1%. The isolates were highly resistant to penicillins, cephalosporins, and monobactams, while very low resistant to carbapenems. Four PFGE profiles were identified:
Hybridization of EcoRI- and HindIII-digested chromosomal DNAs from 41 isolates of Enterococcus faecalis with probes for rRNA genes was performed (ribotyping). The ability of ribotyping to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). With EcoRI, seven ribopatterns (usually differing by only one band) were found, while PFGE had previously shown 25 clearly different patterns plus six related variants. Digestion with HindIII generated a few additional patterns but still failed to differentiate some strains that had very different PFGE patterns. Ribotyping with BscI has also been reported to be inadequate for subspecies strain differentiation (L. M. Hall, B. Duke, M. Guiney, and R. Williams, J. Clin. Microbiol. 30:915-919, 1992). Although ribotyping with other restriction endonucleases may perform better in distinguishing different strains, at present PFGE appears to be superior for strain differentiation. ...
TY - JOUR. T1 - Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments. T2 - A multicenter study. AU - Van Belkum, Alex. AU - Van Leeuwen, Willem. AU - Kaufmann, Mary Elizabeth. AU - Cookson, Barry. AU - Forey, Françoise. AU - Etienne, Jerome. AU - Goering, Richard V.. AU - Tenover, Fred. AU - Steward, Christine. AU - OBrien, Frances. AU - Grubb, Warren. AU - Tassios, Panayotis. AU - Legakis, Nicholas. AU - Morvan, Anne. AU - El Solh, Névine. AU - De Ryck, Raf. AU - Struelens, Marc. AU - Salmenlinna, Saara. AU - Vuopio-Varkila, Jaana. AU - Kooistra, Mirjam. AU - Talens, Adriaan. AU - Witte, Wolfgang. AU - Verbrugh, Henri. PY - 1998/6. Y1 - 1998/6. N2 - Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction ...
Nosocomial Candida albicans infections are a significant problem in neonatal intensive care units (NICUs). We investigated the clonality of C. albicans isolates recovered over an 8-year period from neonates at a NICU. We also validated multilocus sequence typing (MLST) compared with pulsed-field gel electrophoresis (PFGE) for the genotyping of C. albicans strains from the same NICU. A total of 43 clinical isolates (10 blood, 19 urine, and 14 other) were obtained from 43 neonates between 2005 and 2012. Clonal strains were defined as the isolation of two or more strains with identical or similar genotypes as determined with both MLST and PFGE. Using MLST, the 43 isolates yielded 25 diploid sequence types (DSTs) and 10 DSTs were shared by 28 isolates (65.1%). Among the 28 isolates sharing 10 DSTs, isolates from each of seven DSTs had the same or similar PFGE pattern. In addition, two sets of isolates that differed by MLST at only one locus had the same or similar PFGE pattern. Overall, when the MLST and
Electrophoreseis is usually carried out within a matrix or gel made of agarose or polyacrylamide. These gels are chemically inert, so they will interfere little with the molecules. The sample is loaded in the gel;in wells for nucleic acids separation. Agarose is a polysaccharide extracted from seaweed. Agarose gels have a large range of separation depending on the concentration.The higher the concentration the smaller the pore size will be.Polyacrylamide is a cross-linked polymer of acrylamide.To avoid inhibition of polymerization by oxygen, they are poured between glass plates to mage gel slabs.Low concentration of polyacrylamide or less cross linking results in gels with large pores. Standard protein gels are typically composed of two layers, ahe top-most layer called the stacking gel and a lower layer called separating or resolving gel.The stacking layer contains a low percentage of acylamide and low pH , while the acrylamide concentration of the separating gel varies according to the samples ...
Pulsed-field gel electrophoresis (PFGE) was performed with a contour-clamped homogeneous electric field DRII apparatus from Bio-Rad Laboratories (Richmond, Calif.) as described previously (7). The chromosomal DNA was digested overnight with XbaI (GIBCO-BRL, Life Technologies, Gaithersburg, Md.). DNA was electrophoresed in 1.2% SeaKem GTG agarose (FMC) at 6 V/cm for 24 h; the pulse time was increased from 5 to 40 s. Because a single base mutation in the chromosomal DNA of an isolate is sufficient to introduce differences in three fragments in its restriction pattern, isolates with restriction patterns showing the same differences in one to three fragments were considered to belong to the same genotype (23). The PFGE patterns were also analyzed with the computer software Gelcompar for Windows version 3.1b (Applied Math, Kortrijk, Belgium). The PFGE patterns were compared by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages) with the Dice ...
Comprehensive instructions for specimen collection, special requirements, specimen handling, testing methods and turnaround times.
Among 2179 Salmonella isolates obtained during national surveillance for salmonellosis in China from 2005 to 2013, we identified 46 non-H2S-producing strains originating from different sources. The isolates were characterized in terms of antibiotic resistance and genetic variability by pulsed-field gel electrophoresis and multilocus sequence typing. Mutation in the phs operon, which may account for the non-H2S-producing phenotype of the isolated Salmonella strains, was performed in this study. Among isolated non-H2S-producing Salmonella strains, more than 50% were recovered from diarrhea patients, of which H2S-negative S. Gallinarum, S. Typhimurium, S. Choleraesuis and S. Paratyphi A isolates constituted 76%. H2S-negative isolates exhibited a high rate of resistance to ticarcillin, ampicillin, and tetracycline, and eight of them had the multidrug resistance phenotype. Most H2S-negative Salmonella isolates had similar pulsed-field gel electrophoresis profiles and the same sequence type as H2S-positive
Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of|100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make
Recognition of viruses as the most abundant component of aquatic microbial communities has stimulated investigations of the impact of viruses on bacterio- and phytoplankton host communities. From results of field studies to date, it is concluded that in most aquatic environments, a reduction in the number of bacteria on a daily basis is caused by viral infection. However, the modest amount of in situ virus-mediated mortality may be less significant than viral infection serving to maintain clonal diversity in the host communities directly, through gene transmission (i,e,, transduction), and indirectly, by elimination of numerically dominant host species. If the latter mechanism for controlling community diversity prevails, then the overall structure of aquatic viral communities would be expected to change as well over short seasonal and spatial scales, To determine whether this occurs, pulsed-field gel electrophoresis (PFGE) was used to monitor the population dynamics of Chesapeake Bay ...
Fluoroquinolone (FQ)-resistant extraintestinal pathogenic Escherichia coli (FQ(r) ExPEC) strains from phylogenetic group B2 are undergoing epidemic spread. Isolates belonging to phylogenetic group B2 are generally more virulent than other E. coli isolates; therefore, resistance to FQs among group B2 isolates is concerning. Although clonal expansion of sequence type 131 (ST131) is a major factor, the contribution of additional clonal groups has not been quantified. Group B2 FQ(r) ExPEC isolates from humans (n = 250) and dogs (n = 12) in Australia were screened for ST131, a recently recognized and rapidly emerging multidrug-resistant and virulent clonal group that is important in both human and companion animal medicine. Non-ST131 isolates underwent virulence genotyping, PCR-based O typing, partial multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and FQ resistance mechanism analysis. Of 49 non-ST131 isolates (45 human, 4 canine), 49% (24 human, 2 canine) represented ...
Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa ...
Streptococcus pneumoniae plays an important role in causing acute exacerbations in patients with chronic respiratory disease. However, few data are available regarding pneumococcal persistence in adult patients with chronic respiratory diseases. Fifty pneumococci recovered from sputum samples (1995 to 2010) from 13 adult patients with ≥ 3 episodes of acute exacerbation or pneumonia, with the same serotype and pulsed-field gel electrophoresis (PFGE) pattern, were studied. Multilocus sequence typing (MLST) loci, penicillin-binding protein (PBP) genes (pbp2x, pbp1a, pbp2b), and the quinolone-resistant determining regions (QRDRs) of parC, parE, and gyrA were PCR amplified and sequenced. The average time between the first and last episode was 582 days (standard deviation [SD], ± 362). All but two patients received multiple courses of β-lactam treatment, and all persistent strains were resistant to penicillin; however, the PBP sequences were stable over time apart from one variable nucleotide in pbp2x,
Recently, multiresistant Salmonella enterica serovar 1,4,[5],12:i:-, a monophasic variant of S. Typhimurium (1,4,[5],12:i:1,2) emerged, and is now among the most common serovars isolated from humans in many countries. In Greece, monophasic Typhimurium which was recorded for the first time in human isolates in 2007 (0.3% of total isolates), increased sharply thereafter, and since 2009 is the third most frequent serovar. In the present study, 119 S. enterica 1,4,[5],12:i:- strains of human, animal and food origin, isolated during the period between 2006 and 2011, were examined. Strains verified as monophasic Typhimurium variants by polymerase chain reaction (PCR) (97 strains), were further characterised by phenotypic (antibiotic resistance and phage typing) and molecular (pulsed-field gel electrophoresis - PFGE) methods. The results indicate that multiple clones of multiresistant monophasic Typhimurium are circulating in Greece. The most frequently encountered clone in humans and pigs was that of phage
Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil ...
RESULTS Between September 2 and October 22, 2015, 19 surgical patients, including 10 organ transplant recipients and 1 HCW, had positive S. Isangi cultures. Of these case patients and HCW, 13 had gastroenteritis, 2 had bacteremia, 1 had surgical-site infection, and 4 were asymptomatic. Pulsed-field gel electrophoresis (PFGE) showed 89.5% similarity among the isolates in these cases. Isolates with resistant-phenotypes possessed plasmid-mediated CTX-M15 ESBL. A total of 19 case patients were compared with 57 control participants. Case patients had significantly higher odds of exposure to an intraoperative transesophageal (TEE) probe (adjusted odds ratio 9.0; 95% confidence interval, 1.12-72.60; P=.02). Possible cross-transmission occurred in the HCW and 2 patients. Cultures of TEE probes and the environment were negative. The outbreak ended after removal of TEE probes, modification of reprocessing procedures, implementation of strict infection control practices, and enhanced environmental ...
Abstract: To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated ...
Eleven Salmonella spp. isolates with the antigenic type 11:z41:e,n,z15 - not referred to in the 9th edition of the White-Kauffman-Le Minor Scheme - were identified at the National Reference Laboratory for Salmonella in Greece. Their pulsed-field gel electrophoresis profiles were indistinguishable. No apparent epidemiological link has yet been identified; the results of a case-case study are pending.
In article ,3j1dqu$3t1 at mserv1.dl.ac.uk,, MS WF HONG [GEN]53872 ,GEN282E at ccs1.cc.monash.edu.au, writes: , Dear netters, , I am working on genomic mapping of Pseudomonas by pulse field , gel electrophoresis. At this stage, I have a problem in separating a , 173kb doublet. Does any one have suggestions on how to solve this , problem. , , , Thanks in advance. , , , , W. F. Hong , Well, the best way probably is making a gene library and pulling out linking clones hybridizing the library with the 173kb doublet probe.Making the library shouldnt be problem considering the high and reliable packaging efficiency of commercial pack. mix (Stratagene) like Gigapack Good luck. Goran Biukovic , biukovic at olimp.irb.hr ...
Over past 10 years, Rx Biosciences has designed and developed novel modules and technologies to enhance research in protein engineering and genetic recombination. Staff at Rx Biosciences come from diverse and strong backgrounds often with years of research experience. With a state-of-the-art research facility, Rx Biosciences has a up-to-date infrastructure for handling small and large scale projects. Our labs are well equipped with the latest scientific devices including, fully automated Affymetrix platform for micro-array analysis, ABI, Illumina and Beckman sequencers, Pulse Field Gel Electrophoresis Apparatus, ultra-centrifuge, liquid handling platform, cell culture and animal house facilities.. ...
Early genetic studies showed conservation of gene order in the enteric bacteria. Two recent methods using pulsed field gel electrophoresis (PFGE) to determine the physical map of the genome are: (i) partial digestion with the endonuclease I-CeuI, which digests the DNA of bacteria in the rrn operon f …
DI-fusion, le Dépôt institutionnel numérique de lULB, est loutil de référencementde la production scientifique de lULB.Linterface de recherche DI-fusion permet de consulter les publications des chercheurs de lULB et les thèses qui y ont été défendues.
The SageELF is an electrophoresis system that separates DNA or protein samples by size, and then fractionates the whole sample, or section of sample, into 12 fractions. The system is equipped with pulsed-field electrophoresis for resolving large DNA.. Fractionation ranges are estimated and adjusted in software, and fractions are collected in buffer. One sample is fractionated on a single precast agarose cassette, and one or two cassettes may be processed at one time.. Benefits of the SageELF System: ...
This chapter summarizes the recent findings of bacterial genomics and comments on the themes and trends which are emerging. A variety of techniques and methods are available to construct physical maps, and those most commonly employed involve pulsed field gel electrophoresis (PFGE) of macrorestriction fragments generated by digesting intact genomic DNA, immobilized in agarose plugs, with rare-cutting enzymes. Hybridization techniques are often used to construct a map and to deduce the positions of genetic markers. In recent years significant effort has been devoted to developing direct-mapping techniques for large DNA molecules that do not require gel electrophoresis. Among the more promising of these are two new methods known as DNA combing and optical mapping, both of which make use of fluorescence microscopy and image analysis to visualize single DNA molecules. Overall, bacterial genomes range in size from about 0.6 to 9.4 Mb. In a recent review, it was suggested that there may be a relationship
The first time, I used 6ul DNA with 2ul each of enzyme and buffer in a 20ul reaction mix and incubated at 25 degrees (SmaIs optimal temp) overnight, before heat-inactivating, treating with TSAP, and transforming 0.125ul of the mixture into E. coli. The second time, I decreased the amount of DNA in the digest to 2ul, incubated the digest for 4hrs, and used 0.25ul for transformation (didnt bother with the TSAP). I got lots (,50) of colonies both times, although somewhat fewer the second time due to the smaller amount of DNA, and when I do the actual ligation and try the transformation with that, I dont get any more colonies compared to using the equivalent amount of digested vector ...
STROUD WATER RESEARCH CENTER INC - Since 1967, Stroud Water Research Center s scientists and educators have been focused on one thing fresh water. At the heart ...
A total of 486 Listeria monocytogenes isolates originating from 17 Finnish food processing plants (representing meat, poultry, fish, and dairy production) were collected and typed by automated ribotyping using EcoRI as the restriction enzyme. The isolates were divided into 16 different ribotypes (RTs). Some of these isolates (121), representing all EcoRI types and 16 food plants, were subjected to ribotyping with the PvuII enzyme, to pulsed-field gel electrophoresis (PFGE) typing with AscI and SmaI restriction enzymes, and to serotyping with O-antigen antisera. Nineteen ribotypes were generated with PvuII, 42 macrorestriction patterns were generated with AscI and 24 with SmaI, and three serotypes were generated with antisera. When the results were combined, the overall number of RTs was 23, and that of the PFGE types was 46. Thus, the overall discrimination power of PFGE was higher (discrimination index [DI] 0.966) than that of ribotyping (DI 0.906). The most common serotype (90.1% of the isolates) was
In the United States, methicillin-resistant Staphylococcus aureus (MRSA) with the USA300 pulsed-field gel electrophoresis type causes most community-associated MRSA infections and is an increasingly common cause of health care-associated MRSA infections. USA300 probably emerged during the early 1990s. To assess the spatiotemporal diffusion of USA300 MRSA and USA100 MRSA throughout the United States, we systematically reviewed 354 articles for data on 33,543 isolates, of which 8,092 were classified as USA300 and 2,595 as USA100. Using the biomedical literature as a proxy for USA300 prevalence among genotyped MRSA samples, we found that USA300 was isolated during 2000 in several states, including California, Texas, and midwestern states. The geographic mean center of USA300 MRSA then shifted eastward from 2000 to 2013. Analyzing genotyping studies enabled us to track the emergence of a new, successful MRSA type in space and time across the country ...
Staphylococcus aureus (S. aureus) is a major cause of human morbidity and mortality. Strains classified as pulsed-field gel electrophoresis type (PFGE) USA300 a...
The aim of the study was to determine antimicrobial resistance and genotypic characteristics of L. monocytogenes isolated from food of animal origin from different parts of Poland during years 2013-2016. A total of 146 isolates were tested using a microbroth dilution method, whereas virulence genes and molecular serogroups were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods were used to analyze the genotypic relationship of the strains. Altogether, 102 pulsotypes grouped into 7 clusters and 24 sequence types, including 3 new types, were identified. Most of the strains clustered into individual patterns were originated from different food products and were isolated in different geographical regions at various time. L. monocytogenes was mostly resistant to oxacilin (90.4% strains), clindamycin (54.1%) and ceftriaxone (49.3%). A multiresistance patterns, mainly to ceftriaxone, oxacillin together with other antimicrobials, were observed among ...
Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. ...
Objective To determine the penicillin resistance and serotype distribution of Streptococcus pneumoniae strains and to identify clonal relationships of isolates resistant to penicillin by means of pulsed-field gel electrophoresis (PFGE). ...
Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen in both human and veterinary medicine. The importance of companion animals as reservoirs of human infections is currently unknown. The companion animals of 49 MRSA-infected outpatients (cases) were screened for MRSA carriage, and their bacterial isolates were compared with those of the infected patients using Pulsed-Field Gel Electrophoresis (PFGE). Rates of MRSA among the companion animals of MRSA-infected patients were compared to rates of MRSA among companion animals of pet guardians attending a
Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that periodically changes direction. Standard gel electrophoresis techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, it was unable to separate very large molecules of DNA effectively. DNA molecules larger than 15-20 kb migrating through a gel will essentially move together in a size-independent manner. At Columbia University in 1984, David C. Schwartz and Charles Cantor developed a variation on the standard protocol by introducing an alternating voltage gradient to improve the resolution of larger molecules. This technique became known as pulsed-field gel electrophoresis (PFGE). The development of PFGE expanded the range of resolution for DNA fragments by as much as two orders of magnitude. The procedure for this technique is relatively similar to performing a standard gel ...
OBJECTIVE To determine the scope, source, and mode of transmission of a multifacility outbreak of extensively drug-resistant (XDR) Acinetobacter baumannii. DESIGN Outbreak investigation. SETTING AND PARTICIPANTS Residents and patients in skilled nursing facilities, long-term acute-care hospital, and acute-care hospitals. METHODS A case was defined as the incident isolate from clinical or surveillance cultures of XDR Acinetobacter baumannii resistant to imipenem or meropenem and nonsusceptible to all but 1 or 2 antibiotic classes in a patient in an Oregon healthcare facility during January 2012-December 2014. We queried clinical laboratories, reviewed medical records, oversaw patient and environmental surveillance surveys at 2 facilities, and recommended interventions. Pulsed-field gel electrophoresis (PFGE) and molecular analysis were performed. RESULTS We identified 21 cases, highly related by PFGE or healthcare facility exposure. Overall, 17 patients (81%) were admitted to either long-term ...
in Microbial Drug Resistance : Mechanism, Epidemiology, & Disease (2002), 8(3), 193-200. Stenotrophomonas maltophilia is a nosocomial pathogen with an intrinsic broad-spectrum resistance to beta-lactam compounds and other antibacterial agents. It produces two chromosomal beta-lactamases: a ... [more ▼]. Stenotrophomonas maltophilia is a nosocomial pathogen with an intrinsic broad-spectrum resistance to beta-lactam compounds and other antibacterial agents. It produces two chromosomal beta-lactamases: a clavulanic acid-sensitive class A (L2) and a tetrameric carbapenemase (L1 or BlaS). We screened 40 S. maltophilia multidrug-resistant clinical isolates recovered between 1995 and 1998 in the Varese Hospital (Italy) for the presence of the metallo-beta-lactamase. The isolates were investigated by phenotypic profiling (enzymatic activity and antibiotic resistance pattern) and molecular methods such as PCR and pulsed-field gel electrophoresis (PFGE) to reveal intraspecies diversity. For the tested ...
During 2015-2016, we evaluated the performance of whole-genome sequencing (WGS) as a routine typing tool. Its added value for microbiological and epidemiologic surveillance of listeriosis was compared with that for pulsed-field gel electrophoresis (PFGE), the current standard method. A total of 2,743 Listeria monocytogenes isolates collected as part of routine surveillance were characterized in parallel by PFGE and core genome multilocus sequence typing (cgMLST) extracted from WGS. We investigated PFGE and cgMLST clusters containing human isolates. Discrimination of isolates was significantly higher by cgMLST than by PFGE (p<0.001). cgMLST discriminated unrelated isolates that shared identical PFGE profiles and phylogenetically closely related isolates with distinct PFGE profiles. This procedure also refined epidemiologic investigations to include only phylogenetically closely related isolates, improved source identification, and facilitated epidemiologic investigations, enabling identification
A physical map of the chromosome of Neisseria gonorrhoeae FA1090 has been constructed. Digestion of strain FA1090 DNA with NheI, SpeI, BglII, or PacI resulted in a limited number of fragments that were resolved by contour-clamped homogeneous electric field electrophoresis. The estimated genome size was 2,219 kb. To construct the map, probes corresponding to single-copy chromosomal sequences were used in Southern blots of digested DNA separated on pulsed-field gels, to determine how the fragments from different digests overlapped. Some of the probes represented identified gonococcal genes, whereas others were anonymous cloned fragments of strain FA1090 DNA. By using this approach, a macrorestriction map of the strain FA1090 chromosome was assembled, and the locations of various genetic markers on the map were determined. Once the map was completed, the repeated gene families encoding Opa and pilin proteins were mapped. The 11 opa loci of strain FA1090 were distributed over approximately 60% of ...
We observed an increase in bovine-origin S. Typhimurium isolates that were represented by 2 highly similar PFGE patterns, identified as Washington State PulseNet types TYP035 and TYP187 (Table 2). They were clearly distinguishable from phage type DT104 strains isolated during the same time frame (Appendix Figure). To determine whether these XbaI PFGE types were clonal, we characterized 60 S. Typhimurium isolates, including 32 TYP035-TYP187 isolates from bovines and 19 TYP035-TYP187 isolates from humans, by PFGE following digestion of DNA by SpeI. SpeI patterns within each XbaI type showed only minor banding variations. Thirteen isolates that were XbaI-TYP035 had SpeI patterns indistinguishable from those of XbaI-TYP187 isolates. In the cluster analysis of SpeI patterns, the TYP035 and TYP187 isolates XbaI patterns were at least 90.1% similar compared to an overall 83.7% similarity for all S. Typhimurium isolates in the analysis. Characterization of isolates within the XbaI PFGE type TYP035 also ...
Restriction endonuclease patterns generated by Pulsed-Field Gel Electrophoresis (PFGE) were used to compare 96 strains of dairy propionibacteria originating from dairy products, international and indu
This study analyzed 42 strains collected between 2009C2012 from different hospitals in Beyrouth and North Lebanon to raised understand the epidemiology and carbapenem resistance mechanisms in our collection and to compare the robustness of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), repetitive sequence-based PCR (rep-PCR) and is an opportunistic gram negative pathogen involved in a wide quantity of nosocomial infections like ventilator-associated pneumonia, bloodstream, urinary tract, wound and meningitis infections frequently associated with a high rate of mortality and morbidity [1]. 18 were international clones and 8 European or Asian restricted clones. The International clone II was the major clone reported in 34 countries in Europe, Asia, Africa Australia, USA, and South America. To track and monitor these outbreaks, denote strain relatedness and assign an outbreak strain to its corresponding clonal lineage, many typing methods with different intrinsic ...
Figure 1. Dendogram derived from PFGE profiles of a) ApaI and b) AscI macrorestriction, showing pattern similarity among the 22 L. monocytogenes strains of the study. Clusters are indicated on the left side of the Figure as well as a 90 % similarity line. PFGE groups are shown in rectangles on the right side of the figure. The source and other information associated with each strain are also shown.. The analysis of macrorestriction patterns obtained by PFGE-ApaI showed 68 % similarity of the 22 strains studied, distributed in 2 clusters: I and II as shown in Figure 1a. Closely related isolates (greater than 90 % similarity in banding patterns) were assigned a PFGE group. Cluster I had 3 isolates and included clone 001 and a ground beef strain (BS78) with a similarity of 83 %. Cluster II had 19 strains and included the PFGE-ApaI group 1 that grouped 15 strains with 92 % similarity. This group contained 80 % (12/15) of the ground beef strains, the strain isolated in the human sporadic case of 2008 ...
Figure 1. Dendogram derived from PFGE profiles of a) ApaI and b) AscI macrorestriction, showing pattern similarity among the 22 L. monocytogenes strains of the study. Clusters are indicated on the left side of the Figure as well as a 90 % similarity line. PFGE groups are shown in rectangles on the right side of the figure. The source and other information associated with each strain are also shown.. The analysis of macrorestriction patterns obtained by PFGE-ApaI showed 68 % similarity of the 22 strains studied, distributed in 2 clusters: I and II as shown in Figure 1a. Closely related isolates (greater than 90 % similarity in banding patterns) were assigned a PFGE group. Cluster I had 3 isolates and included clone 001 and a ground beef strain (BS78) with a similarity of 83 %. Cluster II had 19 strains and included the PFGE-ApaI group 1 that grouped 15 strains with 92 % similarity. This group contained 80 % (12/15) of the ground beef strains, the strain isolated in the human sporadic case of 2008 ...
TY - JOUR. T1 - Insertion element IS3-based PCR method for subtyping Escherichia coli O157. T2 - H7. AU - Thompson, Curt J.. AU - Daly, Claire. AU - Barrett, Timothy J.. AU - Getchell, Jane P.. AU - Gilchrist, Mary J R. AU - Loeffelholz, Mike J.. PY - 1998/5. Y1 - 1998/5. N2 - An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed- field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single-primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, ...
The RESOLUTION System is based on PathoGenetixs proprietary Genome Sequence Scanning™ (GSS™) technology, which enables pathogen serotype identification and strain typing in just five hours, directly from complex mixtures such as environmental, clinical and enriched food samples. Initially developed to detect bio-threat pathogens in environmental samples under a five-year, $50-million contract through the Department of Homeland Security, the breakthrough GSS technology isolates and analyzes DNA direct from complex mixtures-without the need for a pure culture isolate. The strain type information provided by GSS is comparable in resolution to pulsed field gel electrophoresis (PFGE), one of the current gold standards for pathogen identification ...
Advanced Analytical Technologies Inc AATI has received an RampD 100 Award from RampD Magazine for its FEMTO Pulse automated pulsed-field capillary electrophoresis CE instrument. As the only automated pulsed-field CE system on the market, the FEMTO Pulse won the award for its ability to separate and analyze large nucleic acid fragments span...
Pulsed field gel electrophoresis (PFGE) has provided a very reliable system for separation of DNA fragments greater than 50 kb and has made a significant impact on the analysis of both prokaryotic and eukaryotic genomes. The chapters in this book cover both the theory behind this very important technique and present detailed protocols for many of its major uses.
(2002) Yamaguchi et al. J.Infect.Dis.. A molecular epidemiological analysis was performed to reveal the clonal association of Staphylococcus aureus strains isolated from patients with bullous impetigo. Pulsed-field gel electrophoresis w...
Allele, Antigen, Disease, Electrophoresis, Gene, Identification, Methods, Molecular Typing, Multilocus Sequence Typing, Neisseria, Neisseria Meningitidis, Patients, Pulsed-field Gel Electrophoresis, Strains
Pulsed-field gel electrophoresis proficiency testing trials: Toward european harmonization of the typing of food and clinical strains of listeria monocytogenes ...
In a recent article Epidemiology 1990; 1:421-429 I resurrected some historical criticisms of conventional statistics in non-randomized, non-randomly sampled studies, and suggested some improvements to current practice in response to these criticisms. Here, I propose that some resolution can be achieved by separating data analysis into...
I was officially diagnosed in March 2010. I was dating a man who I thought I would one day marry. I had my first outbreak 3 years into the relationship.