scholarly journals Comparison of Restriction Enzymes for Pulsed-Field Gel Electrophoresis Typing of Moraxella catarrhalis

2013 ◽  
Vol 51 (7) ◽  
pp. 2448-2452 ◽  
Author(s):  
S. Marti ◽  
C. Puig ◽  
A. Domenech ◽  
J. Linares ◽  
C. Ardanuy
1999 ◽  
Vol 45 (4) ◽  
pp. 299-303 ◽  
Author(s):  
K T Nguyen ◽  
E J Hansen ◽  
M A Farinha

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.


1999 ◽  
Vol 37 (6) ◽  
pp. 1927-1931 ◽  
Author(s):  
Samir P. Singh ◽  
Hugh Salamon ◽  
Carol J. Lahti ◽  
Mehran Farid-Moyer ◽  
Peter M. Small

Pulsed-field gel electrophoresis (PFGE) is a powerful molecular biology technique which has provided important insights into the epidemiology and population biology of many pathogens. However, few studies have used PFGE for the molecular epidemiology ofMycobacterium tuberculosis. A laboratory protocol was developed to determine the typeability, stability, and reproducibility of PFGE typing of M. tuberculosis. Formal data-analytical techniques were used to assess the genetic diversity elucidated by PFGE analyses using four separate restriction enzymes and by IS6110 RFLP analyses, as well as to assess the concordance among these typing methods. One hundred epidemiologically characterized clinical isolates of M. tuberculosis were genotyped with four different PFGE enzymes (AseI, DraI,SpeI, and XbaI), as well as by RFLP analysis with IS6110. Identical patterns were found among 34 isolates known to be genetically related, suggesting that the PFGE protocol is robust and reproducible. Among 66 isolates representing population-sampled cases, heterozygosity and information content dependency estimates indicate that all five genotyping systems capture quantitatively similar levels of genetic diversity. Nevertheless, comparisons between PFGE analyses and IS6110 typing reveals that PFGE provided more discrimination among isolates with fewer than five copies of IS6110 and less clustering in isolates with five or more copies. The comparisons confirm the hypothesis that the resolution of IS6110 RFLP genotyping is dependent upon the number of IS6110 elements in the genome of isolates. The general concordance among the results obtained with four independent enzymes suggests that M. tuberculosis is a clonal organism. The availability of a robust genotyping technique largely independent of repetitive elements has implications for the molecular epidemiology of M. tuberculosis.


1999 ◽  
Vol 122 (3) ◽  
pp. 417-422 ◽  
Author(s):  
G. MARTINEZ ◽  
K. AHMED ◽  
C. H. ZHENG ◽  
K. WATANABE ◽  
K. OISHI ◽  
...  

Pulsed field gel electrophoresis (PFGE) of the genomic DNA of Moraxella catarrhalis was done in 172 strains isolated from sputum of patients with respiratory infections in Nagasaki (130 strains), Europe (14 strains), Thailand (6 strains), Uganda (3 strains), Bangladesh (5 strains) and Kuwait (14 strains). Restriction endonuclease with SmaI generated 4–16 DNA fragments ranging from 1000 kb to 24·25 kb and was classified into 31 major groups. It was found that there were wide variations of DNA restriction patterns of strains isolated from the same and different geographical areas. DNA restriction patterns of strains isolated in Nagasaki during the last 12 years showed dynamic changes of the predominant strains in each time period. We conclude from this study that PFGE is a suitable method to document interstrain variation in M. catarrhalis.


1999 ◽  
Vol 37 (2) ◽  
pp. 450-452 ◽  
Author(s):  
Hoang Vu-Thien ◽  
Carole Dulot ◽  
Didier Moissenet ◽  
Brigitte Fauroux ◽  
Antoine Garbarg-Chenon

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for the analysis of 13 Moraxella catarrhalis isolates, 11 successive strains isolated from sputa of five children and 2 isolates obtained the same day from twins, were compared. RAPD and PFGE both yielded nine types from the 13 isolates, showing a chronic colonization with one strain in three patients and a successive colonization with different strains in two patients. The promising results obtained with RAPD should be confirmed with a larger number of strains, but RAPD seems as suitable as PFGE for the typing ofM. catarrhalis.


1995 ◽  
Vol 6 (3) ◽  
pp. 141-144 ◽  
Author(s):  
Susan M Davison ◽  
Donald E Low ◽  
Rene H Cruz ◽  
Danielle Beaulieu ◽  
Shelley R Scriver

Pulsed field gel electrophoresis (pfge) was used to compare 59 strains ofMoraxella catarrhalisto evaluatepfgefor the epidemiological typing of this organism.pfge-generated patterns were compared with those obtained by small fragment restriction enzyme analysis (rea) and species-specific probe hybridization. The strains used in the study were isolated from various geographic locations and included proven epidemiologically related strains.pfgeyielded more unique patterns thandna-dnahybridization – 30 versus 18, respectively – but fewer thanrea, which generated 45 unique patterns. Strains that demonstrated the samereapattern ordna-dnahybridization pattern did not always demonstrate the samepfgepattern. For example, in 23 epidemiologically unrelated strains that shared sixreapatterns,pfgedifferentiated the isolates into 12 patterns. Conversely, strains that demonstrated the samepfgepattern did not always demonstrate the samereapattern or hybridization pattern. For example, in 42 strains that shared 13pfgepatterns,readifferentiated the isolates into 31 patterns anddna-dnahybridization differentiated them into 16 patterns. However, compared withrea,pfgeyielded less complex patterns that were more easily comparable, and compared withdna-dnahybridization,pfgewas technically easier.


2000 ◽  
Vol 46 (11) ◽  
pp. 1082-1086 ◽  
Author(s):  
Joanne B Messick ◽  
Geoffrey Smith ◽  
Linda Berent ◽  
Sandra Cooper

The genome size of Eperythrozoon suis, an unculturable haemotropic mycoplasma, was estimated using pulsed-field gel electrophoresis (PFGE). Gamma irradiation was used to introduce one (on the average) double-strand break in the E. suis Illinois chromosome. Restriction enzymes that cut infrequently were also used to analyze genome size. The size estimate for the full-length genome was 745 kilobases (kb), whereas the size estimates based on the summation of restriction fragments ranged from 730 to 770 kb. The 16S rRNA gene was located on the 120-kb MluI fragment, 128-kb NruI fragment, 25-kb SacII fragment, and 217-kb SalI fragment by Southern blotting.Key words: Eperythrozoon suis, 16S rRNA, Mycoplasma pneumoniae group, pulsed-field gel electrophoresis, genome size.


2000 ◽  
Vol 38 (2) ◽  
pp. 625-629 ◽  
Author(s):  
Hisakazu Yano ◽  
Mitsuko Suetake ◽  
Akio Kuga ◽  
Kazuhiko Irinoda ◽  
Ryoichi Okamoto ◽  
...  

To investigate how bacterial pathogens spread from child to child in a day care center, we monitored six children, two boys and four girls, born between August 1995 and November 1997, attending a day care center and analyzed nasopharyngeal samples from them using pulsed-field gel electrophoresis (PFGE). We obtained nasopharyngeal cultures from all of the affected children and almost all of the unaffected children between September 1998 and March 1999 after some children presented simultaneously with purulent rhinorrhea. Moreover, when a child was found to have acute otitis media, nasopharyngeal secretions from the child were independently cultured during treatment. During this period, 28 isolates of Moraxella catarrhalis, 13 ofStreptococcus pneumoniae, and 4 of Haemophilus influenzae were recovered. PFGE gave 8 patterns for M. catarrhalis, 10 for S. pneumoniae, and 1 for H. influenzae. PFGE patterns demonstrated spread of M. catarrhalis between children. However, each occurrence of clusters of infection with M. catarrhalis lasted 2 to 6 weeks, with a change in PFGE pattern between occurrences of clusters. The M. catarrhalis strain infecting each child also changed. Similarly, the S. pneumoniae strain in each child also changed. In contrast, infection with H. influenzaepersisted for about 3 months in an affected child.


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