scholarly journals Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

2013 ◽  
Vol 79 (12) ◽  
pp. 3856-3859 ◽  
Author(s):  
Zhen Zhang ◽  
Hannamari Hintsa ◽  
Ying Chen ◽  
Hannu Korkeala ◽  
Miia Lindström
Keyword(s):  
Gene Cluster ◽  
Gel Electrophoresis ◽  
Clostridium Botulinum ◽  
Southern Hybridization ◽  
Gene Clusters ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Pulsed Field ◽  
Large Plasmids

ABSTRACTA collection of 36Clostridium botulinumtype E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted tobotEandorfX1in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.

scholarly journals Mobile Genetic Elements Related to the Diffusion of Plasmid-Mediated AmpC β-Lactamases or Carbapenemases from Enterobacteriaceae: Findings from a Multicenter Study in Spain

2015 ◽  
Vol 59 (9) ◽  
pp. 5260-5266 ◽  
Author(s):  
L. Zamorano ◽  
E. Miró ◽  
C. Juan ◽  
L. Gómez ◽  
G. Bou ◽  
...  
Keyword(s):  
Southern Hybridization ◽  
Mobile Genetic Elements ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Genetic Elements ◽  
Chromosomal Origin ◽  
Class 1 ◽  
Rapid Dissemination ◽  
Large Plasmids ◽  
Genetic Context

ABSTRACTWe examined the genetic context of 74 acquiredampCgenes and 17 carbapenemase genes from 85 of 640Enterobacteriaceaeisolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74blaAmpCgenes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquiredampCgenes. TheblaCMY-2-like genes were associated with ISEcp1; the surroundingblaDHAgenes were similar toKlebsiella pneumoniaeplasmid pTN60013 associated with IS26and thepspandsapoperons; and theblaACC-1genes were associated with IS26elements inserted into ISEcp1. All of the carbapenemase genes (blaVIM-1,blaIMP-22, andblaIMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination ofampCgenes amongEnterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study.

Construction of a genomic map ofMoraxella (Branhamella) catarrhalisATCC 25238 and physical mapping of virulence-associated genes

1999 ◽  
Vol 45 (4) ◽  
pp. 299-303 ◽  
Author(s):  
K T Nguyen ◽  
E J Hansen ◽  
M A Farinha
Keyword(s):  
Gel Electrophoresis ◽  
Moraxella Catarrhalis ◽  
Southern Hybridization ◽  
Physical Map ◽  
Outer Membrane Proteins ◽  
Restriction Enzymes ◽  
Pulsed Field Gel Electrophoresis ◽  
Respiratory Pathogens ◽  
Circular Chromosome ◽  
Pulsed Field

A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis. Macrorestriction analyses of the genome of M. catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively. The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations. Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome. The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease. A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase.Key words: Moraxella catarrhalis, physical map, genome analysis, pulsed-field gel electrophoresis, virulence.

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

2006 ◽  
Vol 52 (9) ◽  
pp. 857-867 ◽  
Author(s):  
Ellen L Dally ◽  
Thereza S.L Barros ◽  
Yan Zhao ◽  
ShaoPing Lin ◽  
Bruce A Roe ◽  
...  
Keyword(s):  
Genetic Map ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Enzyme Analysis ◽  
Two Dimensional ◽  
Pulsed Field ◽  
Spiroplasma Kunkelii ◽  
Corn Stunt ◽  
Physical And Genetic Map

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.Key words: Spiroplasma kunkelii CR2-3x, corn stunt spiroplasma, mollicutes, genome mapping, two-dimensional pulsed-field gel electrophoresis.

scholarly journals Comparison of Virulence Gene Identification, Ribosomal Spacer PCR, and Pulsed-Field Gel Electrophoresis for Typing of Staphylococcus aureus Strains Isolated from Cases of Subclinical Bovine Mastitis in the United States

2016 ◽  
Vol 54 (7) ◽  
pp. 1871-1876 ◽  
Author(s):  
Pamela R. F. Adkins ◽  
John R. Middleton ◽  
Lawrence K. Fox
Keyword(s):  
United States ◽  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Virulence Gene ◽  
The United States ◽  
Pulsed Field Gel Electrophoresis ◽  
Gene Identification ◽  
Content Type ◽  
Pulsed Field

Staphylococcus aureusis one of the most important pathogens causing contagious mastitis in dairy cattle worldwide. The objectives of this study were to determine if recently describedS. aureusgenotype B was present among previously characterized isolates from cases of bovine intramammary infection in the United States and to compare pulsed-field gel electrophoresis (PFGE) to the combination of ribosomal spacer PCR (RS-PCR) and virulence gene identification for typing ofS. aureusstrains. The hypothesis was that isolates that were previously characterized as contagious would be identified as genotype B and that the results of the two strain-typing methods would be comparable. Isolates were selected from a collection ofS. aureusisolates from eight dairy farms. Mammary quarter milk somatic cell count (SCC) andN-acetyl-β-d-gluconaminidase (NAGase) activity data were known and used to evaluate strain pathogenicity. RS-PCR was performed with conventional gel electrophoresis, and PCR was used for toxin gene identification. RS-PCR patterns were associated with a specific virulence gene pattern, as previously reported. Five RS-PCR banding patterns were identified. None of the isolates were characterized as genotype B. No association between RS-PCR types and milk SCC was found; however, NAGase activity was significantly higher in milk from mammary glands infected with RS-PCR banding type 1 (RSP type 1) than in milk from those infected with RSP type 2. The discriminatory power values were 1.0 and 0.46 for PFGE and RS-PCR, respectively. These data suggest that genotype B may have a limited geographic distribution and that PFGE is more discriminatory than RS-PCR performed with conventional gel electrophoresis for typing ofS. aureusisolates of bovine origin.

scholarly journals Pulsed-Field Gel Electrophoresis and PCR Characterization of Environmental Vibrio parahaemolyticus Strains of Different Origins

2011 ◽  
Vol 77 (17) ◽  
pp. 6301-6304 ◽  
Author(s):  
E. Suffredini ◽  
C. Lopez-Joven ◽  
L. Maddalena ◽  
L. Croci ◽  
A. Roque
Keyword(s):  
Gel Electrophoresis ◽  
Vibrio Parahaemolyticus ◽  
Genetic Relationships ◽  
Pulsed Field Gel Electrophoresis ◽  
Environmental Isolates ◽  
Content Type ◽  
Pulsed Field ◽  
Intraspecies Variability ◽  
Different Origins

ABSTRACTThe present study used pulsed-field gel electrophoresis (PFGE) characterization to examine the intraspecies variability and genetic relationships among environmental isolates ofVibrio parahaemolyticusfrom different European countries. This is first study performed on environmentalV. parahaemolyticusthat included more than one European country.

scholarly journals Metallo-β-Lactamases in ClinicalPseudomonas Isolates in Taiwan and Identification of VIM-3, a Novel Variant of the VIM-2 Enzyme

2001 ◽  
Vol 45 (8) ◽  
pp. 2224-2228 ◽  
Author(s):  
Jing-Jou Yan ◽  
Po-Ren Hsueh ◽  
Wen-Chien Ko ◽  
Kwen-Tay Luh ◽  
Shu-Huei Tsai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Novel Variant ◽  
Pcr Assays ◽  
Positive Results

ABSTRACT A total of 209 clinical isolates of Pseudomonas (193Pseudomonas aeruginosa, 10 P. putida, 4P. stutzeri, and 2 P. fluorescensisolates) with reduced susceptibilities to imipenem and/or ceftazidime were subjected to PCR assays with primers specific forbla IMP-1, bla IMP-2,bla VIM-1, and bla VIM-2and sequence analysis to identify the metallo-β-lactamases (MBLs) prevalent among these organisms in Taiwan; and 21 isolates gave positive results. Five isolates including two P. putida and three P. stutzeri isolates were found to carrybla IMP-1, and six isolates including fiveP. putida and one P. stutzeri isolates harboredbla VIM-2. The remaining 10 isolates wereP. aeruginosa, and all were found to carry a novel variant of bla VIM-2, designatedbla VIM-3. There are only two nucleotide differences between bla VIM-2 andbla VIM-3, leading to two amino acid alterations. Our findings indicate that VIM-2 and its variant have become the most prevalent metalloenzymes in Pseudomonas in Taiwan. Southern hybridization with thebla VIM-2-, bla VIM-3-, and bla IMP-1 -specific probes revealed that only two VIM-2-producing P. putida isolates appeared to carry the MBL gene on plasmids. Pulsed-field gel electrophoresis showed that six VIM-3-producing P. aeruginosa isolates and two IMP-1-producing P. stutzeri isolates were genetically related, suggesting that the spread of these MBL genes in Taiwan could be due to clonal dissemination as well as genetic exchange between different clones.

scholarly journals A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP.

1988 ◽  
Vol 167 (2) ◽  
pp. 664-669 ◽  
Author(s):  
J Rey-Campos ◽  
P Rubinstein ◽  
S Rodriguez de Cordoba
Keyword(s):  
Gene Cluster ◽  
Complement Activation ◽  
Gel Electrophoresis ◽  
Physical Map ◽  
Pulsed Field Gel Electrophoresis ◽  
Complement Components ◽  
Pulsed Field ◽  
Human Genes ◽  
Tight Linkage ◽  
Genes Encoding

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster.

scholarly journals Prevalence of Clostridium botulinum in Finnish Trout Farms: Pulsed-Field Gel Electrophoresis Typing Reveals Extensive Genetic Diversity among Type E Isolates

1998 ◽  
Vol 64 (11) ◽  
pp. 4161-4167 ◽  
Author(s):  
Sebastian Hielm ◽  
Johanna Björkroth ◽  
Eija Hyytiä ◽  
Hannu Korkeala
Keyword(s):  
Genetic Diversity ◽  
Gel Electrophoresis ◽  
Clostridium Botulinum ◽  
Pulsed Field Gel Electrophoresis ◽  
Most Probable Number ◽  
Probable Number ◽  
Pulsed Field ◽  
Pfge Typing ◽  
Specific Pcr ◽  
Trout Farms

ABSTRACT The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay. C. botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C. botulinum type E spores kg−1, respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C. botulinum type E was high. A numerical analysis of SmaI-XmaI macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters.

scholarly journals Genomic Complexity among Strains of the Facultative Photoheterotrophic Bacterium Rhodobacter sphaeroides

1999 ◽  
Vol 181 (5) ◽  
pp. 1684-1688 ◽  
Author(s):  
Kirsten Siedenburg Nereng ◽  
Samuel Kaplan
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Restriction Endonucleases ◽  
Pulsed Field ◽  
Genomic Complexity ◽  
Genome Complexity

ABSTRACT Pulsed-field gel electrophoresis following the use of rare cutting restriction endonucleases together with Southern hybridization, using markers distributed on chromosomes I and II of Rhodobacter sphaeroides 2.4.1, has been used to examine approximately 25 strains of R. sphaeroides in an effort to assess the occurrence of genome complexity in these strains. The results suggest that genome complexity is widespread and is accompanied by substantial genomic heterogeneity.

scholarly journals Intermediately virulent Rhodococcus equi isolates from pigs in Slovenia: discovery of new plasmid types and assessment of genetic diversity by pulsed-field gel electrophoresis

2009 ◽  
Vol 54 (No. 3) ◽  
pp. 111-117 ◽  
Author(s):  
M. Pate ◽  
M. Ocepek ◽  
I. Zdovc ◽  
C. Minato ◽  
Y. Ohtsu ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Gel Electrophoresis ◽  
Detailed Comparison ◽  
Rhodococcus Equi ◽  
Pulsed Field Gel Electrophoresis ◽  
High Genetic Diversity ◽  
Pulsed Field ◽  
Large Plasmids ◽  
Multiple Strains ◽  
Pfge Profiles

The presence of large plasmids in 30 <I>Rhodococcus equi</I> strains isolated from pig lymph nodes with granulomatous changes was investigated. Plasmid DNAs were isolated and digested with the restriction endonucleases <I>Bam</I>HI, <I>Eco</I>RI, <I>Eco</I>T22I and <I>Hind</I>III for detailed comparison and estimation of plasmid sizes. A total of nine isolates were identified as intermediately virulent (VapB-positive), harbouring large plasmids of type 5 (<I>n</I> = 5) and four new variants that we tentatively designated as type 19 (<I>n</I> = 1), 20 (<I>n</I> = 1), 21 (<I>n</I> = 1) and 24 (<I>n</I> = 1). All isolates were subjected to genotyping with pulsed-field gel electrophoresis (PFGE). High genetic diversity was observed: 21 distinct genotypes were detected; five were found in multiple isolates and the others were unique. Isolates of the same plasmid type exhibited different PFGE profiles and vice versa. In a few cases, multiple strains from certain farms were analysed, the majority of which exhibited diverse PFGE profiles. Our findings demonstrate the presence of a wide variety of <I>R. equi</I> strains even in small confined environments such as farms. This is the first molecular epidemiology study of intermediately virulent <I>R. equi</I> isolates from Slovenian pigs.

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