scholarly journals Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis

2007 ◽  
Vol 73 (15) ◽  
pp. 4984-4995 ◽  
Author(s):  
Gerald D. Baldridge ◽  
Nicole Y. Burkhardt ◽  
Roderick F. Felsheim ◽  
Timothy J. Kurtti ◽  
Ulrike G. Munderloh
Keyword(s):  
Southern Blot ◽  
Gel Electrophoresis ◽  
Fluorescent Protein ◽  
Pulsed Field Gel Electrophoresis ◽  
Marker Genes ◽  
Rickettsia Felis ◽  
Pulsed Field ◽  
Rickettsia Monacensis ◽  
Obligate Intracellular Bacteria ◽  
Green Fluorescent

ABSTRACT Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.

Molecular Characterization of Escherichia coli O157:H7 Hide Contamination Routes: Feedlot to Harvest

2006 ◽  
Vol 69 (6) ◽  
pp. 1240-1247 ◽  
Author(s):  
K. D. CHILDS ◽  
C. A. SIMPSON ◽  
W. WARREN-SERNA ◽  
G. BELLENGER ◽  
B. CENTRELLA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Gel Electrophoresis ◽  
Side Wall ◽  
Pulsed Field Gel Electrophoresis ◽  
Marker Genes ◽  
Escherichia Coli O157 ◽  
Pulsed Field ◽  
E Coli ◽  
Side Rail

This study was conducted to identify the origin of Escherichia coli O157:H7 contamination on steer hides at the time of harvest. Samples were collected from the feedlot, transport trailers, and packing plant holding pens and from the colons and hides of feedlot steers. A total of 50 hide samples were positive for E. coli O157:H7 in two geographical locations: the Midwest (25 positive hides) and Southwest (25 positive hides). Hide samples were screened, and the presence of E. coli O157: H7 was confirmed. E. coli O157:H7 isolates were fingerprinted by pulsed-field gel electrophoresis and subjected to multiplex PCR procedures for amplification of E. coli O157:H7 genes stx1, stx2, eaeA, fliC, rfbEO157, and hlyA. Feedlot water trough, pen floor, feed bunk, loading chute, truck trailer side wall and floor, packing plant holding pen floor and side rail, and packing plant cattle drinking water samples were positive for E. coli O157:H7. Pulsed-field gel electrophoresis banding patterns were analyzed after classifying isolates according to the marker genes present and according to packing plant. In this study, hide samples positive for E. coli O157:H7 were traced to other E. coli O157:H7–positive hide, colon, feedlot pen floor fecal, packing plant holding pen drinking water, and transport trailer side wall samples. Links were found between packing plant side rails, feedlot loading chutes, and feedlot pens and between truck trailer, different feedlots, and colons of multiple cattle. This study is the first in which genotypic matches have been made between E. coli O157:H7 isolates obtained from transport trailer side walls and those from cattle hide samples within the packing plant.

scholarly journals Determination of Wolbachia Genome Size by Pulsed-Field Gel Electrophoresis

2001 ◽  
Vol 183 (7) ◽  
pp. 2219-2225 ◽  
Author(s):  
Ling V. Sun ◽  
Jeremy M. Foster ◽  
George Tzertzinis ◽  
Midori Ono ◽  
Claudio Bandi ◽  
...  
Keyword(s):  
Genome Size ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Obligate Intracellular ◽  
Before And After ◽  
Wolbachia Genome ◽  
Size Variability ◽  
Obligate Intracellular Bacteria

ABSTRACT Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI,ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes ofwMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.

scholarly journals Evaluation of Genetic Markers and Molecular Typing Methods for Prediction of Sources of Campylobacter jejuni and C. coli Infections

2007 ◽  
Vol 73 (5) ◽  
pp. 1683-1685 ◽  
Author(s):  
Rauni Kärenlampi ◽  
Hilpi Rautelin ◽  
Marja-Liisa Hänninen
Keyword(s):  
Genetic Markers ◽  
Campylobacter Jejuni ◽  
Molecular Typing ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Marker Genes ◽  
Pulsed Field ◽  
Typing Methods

ABSTRACT Campylobacter jejuni and C. coli isolates from poultry, cattle, and humans were studied using pulsed-field gel electrophoresis (PFGE) and PCR of candidate livestock-associated marker genes. Human isolates showed 5.7 and 61% overlap with cattle and poultry isolates, respectively, by use of PFGE. No unambiguous association was found between marker genes (the Cj1321 and Cj1324 genes) and livestock-associated isolates.

scholarly journals Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion inChlamydia trachomatisand Can Reverse Cassette-Induced Polar Effects

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
G. Keb ◽  
R. Hayman ◽  
K. A. Fields
Keyword(s):  
Gene Deletion ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Selection Marker ◽  
Marker Genes ◽  
Content Type ◽  
Allelic Exchange ◽  
Conditional Expression ◽  
Obligate Intracellular Bacteria ◽  
Green Fluorescent

ABSTRACTAs obligate intracellular bacteria,Chlamydiaspp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion ofChlamydia trachomatistmeAnegatively impacts the expression oftmeB. We have adapted FRAEM technology by employing agfp-blacassette flanked byloxPsites. Conditional expression of Cre recombinase inChlamydiatmeAcontaining a floxed cassette resulted in deletion of the marker and restoration oftmeBexpression.IMPORTANCEC. trachomatisinfections represent a significant burden to human health. The ability to genetically manipulateChlamydiaspp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation inChlamydiaspp. requires further development, including mechanisms to generate markerless gene disruption. We leveraged a stepwise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient, and the removal of extraneous elements resulted in the reversal of a negative polar effect on a downstream gene. This technique facilitates a more direct assessment of gene function and adds to theChlamydiamolecular toolbox by facilitating the deletion of genes within operons.

scholarly journals Emergence of mcr-1-Harboring Salmonella enterica Serovar Sinstorf Type ST155 Isolated From Patients With Diarrhea in Jiangsu, China

2021 ◽  
Vol 12 ◽  
Author(s):  
Guoye Liu ◽  
Huimin Qian ◽  
Jingwen Lv ◽  
Benshun Tian ◽  
Changjun Bao ◽  
...  
Keyword(s):  
Southern Blot ◽  
Gel Electrophoresis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Sequencing Analysis ◽  
Colistin Resistance ◽  
Pulsed Field ◽  
Extended Spectrum Beta Lactamase ◽  
Pcr Method

Background: This study analyzed the antimicrobial resistance phenotypes and mechanisms of quinolone, cephalosporins, and colistin resistance in nontyphoidal Salmonella from patients with diarrhea in Jiangsu, China.Methods: A total of 741 nontyphoidal Salmonella isolates were collected from hospitals in major cities of Jiangsu Province, China between 2016 and 2017. Their susceptibility to commonly used antibiotics was evaluated by broth micro-dilution and sequencing analysis of resistance genes screened by a PCR method. For mcr-1 positive isolates, genetic relationship study was carried out by pulsed-field gel electrophoresis and multiloci sequence typing analysis. The transferability of these plasmids was measured with conjugation experiments and the genetic locations of mcr-1 were analyzed by pulsed-field gel electrophoresis profiles of S1-digested genomic DNA and subsequent Southern blot hybridization.Results: Among 741 nontyphoidal Salmonella isolates, the most common serotypes identified were S. Typhimurium (n=257, 34.7%) and S. Enteritidis (n=127, 17.1%), and the isolates showed 21.7, 20.6, and 5.0% resistance to cephalosporins, ciprofloxacin, and colistin, respectively. Among the 335 nalidixic acid-resistant Salmonella, 213 (63.6%) and 45 (13.4%) had at least one mutation in gyrA and parC. Among the plasmid-borne resistance, qnrS1 (85; 41.9%) and aac(6')-Ib-cr4 (75; 36.9%) were the most common quinolone resistance (PMQR) genes, while blaCTX-M-14 (n=35) and blaCTX-M-55 (n=46) were found to be dominant extended-spectrum beta-lactamase (ESBL) genes in nontyphoidal Salmonella. In addition, eight mcr-1-harboring strains were detected since 2016 and they were predominate in children under the age of 7years. Conjugation assays showed the donor Salmonella strain has functional and transferable colistin resistance and Southern blot hybridization revealed that mcr-1 was located in a high molecular weight plasmid.Conclusion: In nontyphoidal Salmonella, there is a rapidly increasing trend of colistin resistance and this is the first report of patients harboring mcr-1-positive Salmonella with a new ST type ST155 and new serotype S. Sinstorf. These findings demonstrate the necessity for cautious use and the continuous monitoring of colistin in clinical applications.

scholarly journals Use of Pulsed-Field Gel Electrophoresis to Determine the Source of Methicillin-Resistant Staphylococcus aureus Bacteremia

2021 ◽  
Vol 13 (3) ◽  
pp. 602-610
Author(s):  
Eugene Y. H. Yeung ◽  
Ivan Gorn
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Epidemiological Studies ◽  
Pelvic Trauma ◽  
Pulsed Field Gel Electrophoresis ◽  
Bacterial Strains ◽  
Methicillin Resistant ◽  
Pulsed Field ◽  
Clinical Cases

Pulsed-field gel electrophoresis (PFGE) has historically been considered the gold standard in fingerprinting bacterial strains in epidemiological studies and outbreak investigations; little is known regarding its use in individual clinical cases. The current study detailed two clinical cases in which PFGE helped to determine the source of their methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Patient A was found to have MRSA bacteremia after trauma in her pelvic area. MRSA was also found in her groin but not in her nostril and rectum. PFGE was performed that showed variable bands of her MRSA isolates from blood and groin, suggestive of different strains of MRSA. Her MRSA bacteremia was determined to be unrelated to her pelvic trauma. Patient B was found to have MRSA bacteremia after colonoscopy. MRSA was also found in his nostril and rectum. PFGE was performed that showed variable bands of his MRSA isolates from blood and rectum but identical bands of MRSA isolates from his blood and nostril. His MRSA bacteremia was determined to be unrelated to his colonoscopy procedure. The current study demonstrates the use of PFGE to rule out the source of bacteremia in individual clinical cases.

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