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Chapter 2 : R Plasmids and Antibiotic Resistances

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Abstract:

It has been estimated that at least 356 extra-chromosomal elements can introduce genetic information into , an amount of exogenously contributed DNA, in the form of plasmids, equal to or greater than the DNA content of the chromosome. The mole fraction G+C values would provide a clue to their origin(s) since the chromosome has a G+C ratio of 0.50, in comparison with a G+C ratio of 0.38 for the chromosome. The author and his coworkers found mole fraction G+C values of 0.39 and 0.40 for plasmids RSF007 and RSF0885, respectively, values that are closer to those for than . To visualize in more detail the exact nature of the TnA sequence in both plasmids, they heteroduplexed each plasmid with R648, a replicon containing the entire ampicillin transposon with the added feature of one EcoRI cleavage site that served to orient TnA in heteroduplex analyses. The incidence of R-plasmid-mediated drug resistance among clinical isolates is most evident in highly selective environments such as intensive care units and burn wards, where the daily administration of high-potency antibiotics is often a necessity. The identities of two conjugative R plasmids isolated from bacterial strains from two gram-negative genera originating in a hospital burn unit were proved, by a variety of molecular techniques. Drug-resistant strains cause otitis media meningitis among children in day-care centers, and in hospitals, clinicians grapple with multidrug-resistant and species.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2

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Chromosomal DNA
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Conjugative Plasmids
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Figures

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Figure 1

Ethidium bromide-CsCl gradient of a cleared lysate of G32(RSF007) grown in minimal medium in the presence of [H]thymidine.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
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Figure 2

Electron micrograph of ampicillin-resistant plasmid RSF007. CCC, covalently closed circular form; OC, open circular form. Final magnification, x 13,874.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
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Figure 3

Heteroduplexes of RSF007 and RSF0885 plasmid DNA with RI-cut R648 plasmid DNA. (A) RI endonuclease-cut single-stranded molecule of R648. The Tn region is marked by the inverted repeats that reanneal in a single-stranded molecule, forming a double-stranded region (arrow). (B) Heteroduplex of RSF007 and RI-cut R648. (C) Single- and double-stranded molecules of RSF0885. Note the absence of an inverted repeat nucleotide base sequence in the single-stranded molecule. (D) Heteroduplex of RSF0885 and RI-cut R648. SS, single-stranded DNA; DS, double-stranded DNA; IR, inverted repeat nucleotide base sequence.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
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Figure 4

(A). Heteroduplex of RSF007 and pUB701 plasmid DNA. (B) Diagram of RSF007/pUB701 heteroduplex. Thick line, double-stranded DNA; thin line, single-stranded DNA; Ap, ampicillin resistance transposon Tn; Tc, tetracycline resistance transposon (Tn); IR, inverted repeat sequence.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
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Figure 5

Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of penicillin-susceptible (Pen) and penicillin-resistant (Pen) strains. Lanes: A, strain KH45 (Pen) containing the 24.5 x 10-Da cryptic plasmid; B, strain F62 (Pen) containing the 2.6 x 10-Da cryptic plasmid; C, strain CDC66 (Pen), Far East isolate; D, strain CDC67 (Pen), Far East isolate; E, spontaneous Pen derivative of strain CDC01; F, strain CDC01 (Pen), from a case contact of a patient infected in the Far East; G, strain IPL (Pen) from London, England; H, standard plasmid DNAs ranging in size from 62 x 10 (uppermost band) to 1.9 x 10 (lowest band) Da. OC refers to the open circular form of the 2.6 x 10-Da cryptic plasmid. Numbers indicate molecular masses (in megadaltons). CHR indicates chromosomal DNA.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
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Figure 6

Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of tobramycin-resistant (TOB) and tobramycin-susceptible strains. Plasmid pIE098 is a 64-MDa conjugative R plasmid specifying resistances to tobramycin, kanamycin, neomycin, streptomycin, and ampicillin. Lanes: A, 185(R); B, TOB clinical isolate; C and D, 185(pIE098), derived from mating with the TOB strain; E, TOB clinical isolate; F and G, 185(pIE098), derived from mating with the TOB isolate; H, purified standard plasmids for molecular weight determinations. Numbers indicate megadaltons. CHR. DNA, chromosomal DNA.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
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Tables

Generic image for table
Table 1

Hybridization between H-labeled RSF0885 and RSF1030 plasmid DNA and whole-cell DNA

The degree of DNA-DNA duplex formation was assayed by the S1 endonuclease method ( ). The actual extent of binding of H-labeled RSF0885 and RSF1030 plasmid DNA with whole-cell (RSF0885) and (RSF1030) DNA was approximately 87%. All other reactions were normalized to these values taken as 100%. Each value shown is the average of three separate reactions.

NT, not tested.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
Generic image for table
Table 2

Properties of selected, naturally occurring, large-molecular-weight R plasmids

Ap, ampicillin; Tc, tetracycline; CAM, chloramphenicol acetyltransferase.

DK, Denmark; NL, Netherlands; FRG, West Germany; UK, United Kingdom; USA, United States.

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2
Generic image for table
Table 3

Molecular characteristics of small β-lactamase-specifying plasmids found in and species

Citation: Elwell L. 1994. R Plasmids and Antibiotic Resistances, p 17-41. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch2

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