Rapid Method for Screening Large Numbers of
            <i>Escherichia coli</i>
            Colonies for Production of Plasmid-Mediated β-Lactamases

Wolfson, John S.; Hooper, David C.; Swartz, Morton N.; Swartz, M D; McHugh, Gail

doi:10.1128/aac.23.2.308

Cited by 11 publications

(11 citation statements)

References 27 publications

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“…The presence of plasmid pMG110 in bacterial colonies growing on agar was determined by a filter paper assay that detects plasmidmediated 1-lactamase production (27,28). For all experiments described here, colonies were spot checked for pMG110-specified resistances to ampicillin, chloramphenicol, and Tc by replica plating; all findings agreed with those from the filter paper assay.…”

supporting

confidence: 55%

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Elimination of plasmid pMG110 from Escherichia coli by novobiocin and other inhibitors of DNA gyrase

Hooper¹,

Wolfson²,

McHugh³

et al. 1984

Antimicrob Agents Chemother

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The ability of novobiocin to eliminate (cure) the wild-type plasmid pMG110 from Escherichia coli has been compared with that of other inhibitors of the gyrase B subunit and of the gyrase A subunit. Novobiocin eliminated pMG110, producing over 99% plasmid loss at concentrations two-to eightfold below the MIC for bacterial growth. Structurally related compounds (clorobiocin, coumermycin Al, isobutyryl novenamine, and decarbamyl novobiocin) varied in their ability to eliminate pMG110. Higher concentrations of drugs were required to eliminate pMG110 from a gyrB(Cour) strain, implicating DNA gyrase in the curing phenomenon. For these drugs, the ratio of the concentration effecting maximal plasmid elimination to the MIC varied from 0.16 to 1.1, indicating that curing cannot be explained simply by inhibition of a pool of DNA gyrase equally available for replication of the bacterial chromosome and the plasmid DNA molecule. Inhibitors of the gyrase A subunit, nalidixic acid and oxolinic acid, eliminated pMG10 only to variable low levels. The differences in the ability of the gyrase A and B subunit antagonists to eliminate plasmids are discussed.

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supporting

confidence: 55%

“…Occasionally, some colonies appeared heterogeneous (either sectors or outer concentric zones appearing negative for P-lactamase). These colonies were scored as plasmid-containing because a fraction of cells within the colony still contained plasmid (28).…”

mentioning

confidence: 99%

Elimination of plasmid pMG110 from Escherichia coli by novobiocin and other inhibitors of DNA gyrase

Hooper¹,

Wolfson²,

McHugh³

et al. 1984

Antimicrob Agents Chemother

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“…The second method used to identify ␤-lactamase-positive transformants employed a chromogenic penicillinase filter assay (43). Colonies representing 5 ϫ 10 4 transformants were screened after transfer to the penicillinase assay filters.…”

Section: Resultsmentioning

confidence: 99%

“…A chromogenic penicillin-bromocresol purple assay was performed on colonies transferred to filters as previously described (28,43). Colonies producing penicillinase activity exhibit a bright yellow color, while colonies that do not are purple.…”

Section: Methodsmentioning

confidence: 99%

Expression of the AsbA1, OXA-12, and AsbM1 beta-lactamases in Aeromonas jandaei AER 14 is coordinated by a two-component regulon

Alksne

Rasmussen

1997

J Bacteriol

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Aeromonas jandaei AER 14 (formerly Aeromonas sobria AER 14) expresses three inducible ␤-lactamases, AsbA1, OXA-12 (AsbB1), and AsbM1. Mutant strains that constitutively overexpress all three enzymes simultaneously, suggesting that they share a common regulatory pathway, have been isolated. Detectable expression of the cloned genes for AsbA1 and OXA-12 in some Escherichia coli K-12 laboratory strains is achieved only in the presence of a blp mutation. These mutations map to the cre operon at 0 min, which encodes a classical two-component regulatory system of unknown function. Two regulatory elements from A. jandaei which permit high-level constitutive expression of OXA-12 in E. coli were cloned. Both loci encode proteins with characteristics of response regulator proteins of two-component regulatory systems. One of these loci, designated blrA, bestowed constitutive expression of all three ␤-lactamases in A. jandaei AER 14 when present on a multicopy plasmid, confirming its role in the regulatory pathway of ␤-lactamase production in this organism.Aeromonas jandaei AER 14 (formerly Aeromonas sobria AER 14 [5]) expresses three chromosomally encoded, inducible ␤-lactamases: a cephalosporin-hydrolyzing enzyme, AsbA1; a cloxacillin-hydrolyzing enzyme, OXA-12 (AsbB1); and a carbapenem-hydrolyzing metalloenzyme, AsbM1 (12,29,45). The three enzymes each exhibit ␤-lactam hydrolysis selectivity with minimal substrate overlap among them. Selection for resistance to the noninducing ␤-lactam cefuroxime identified a mutation that results in constitutive expression of all three enzymes, indicating that they are coordinately regulated (12,29,45). Inducible expression of the chromosomally encoded class C ␤-lactamases of gram-negative bacteria is regulated by a highly conserved and well-described AmpR repression system (15-21); however, little is known about the regulation of other classes of chromosomally encoded ␤-lactamases such as the carbapenem-hydrolyzing metalloenzymes. Mechanisms for the coordinate regulation of more than one inducible ␤-lactamase have not been previously described. Sequence analysis of the promoter regions of the two cloned ␤-lactamase genes asbA1 and asbB1, encoding AsbA1 and OXA-12, respectively, reveals sequence similarities that may be involved in coregulation; however, no ampR-like regulatory gene has been identified (29).In some laboratory strains of Escherichia coli the genes encoding AsbA1 and OXA-12 are expressed at high levels only if a blp mutation, mapping to the cre operon, is present (reference 29 and unpublished data). The cre operon encodes a classical two-component regulatory system of unidentified function (1, 40; for reviews, see references 38 and 39). While the cre sensor kinase component, CreC, has been demonstrated to cross regulate expression of the Pho regulatory machinery in a phoR background, the signals and targets of the Cre system have not been identified. The finding that components of the cre regulatory system affect the expression of the ␤-lactamases in E. coli prompted a se...

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“…Production of plasmid-mediated 1-lactamase was screened by a rapid method using filter paper impregnated with benzylpenicillin and bromocresol purple as a pH indicator (25 Reactions were performed in a DNA Thermal Cycler (Perkin-Elmer Cetus) with 10 mM Tris-hydrochloride (pH 8.3)-50 mM KCl-2 mM MgCl2, 250 ,uM deoxynucleoside triphosphates, 2.5 U of Taq DNA polymerase, 10 ,ul of 10 ,uM stock of each oligonucleotide primer, and the bacterial DNA (final volume, 100 ,ul). Thirty cycles with the following temperature profiles were used for each reaction: 95°C, 1 min; 60°C, 1 min; and 72°C, 2 min.…”

Section: Methodsmentioning

confidence: 99%

Limitations of plasmid complementation test for determination of quinolone resistance due to changes in the gyrase A protein and identification of conditional quinolone resistance locus

Soussy

Wolfson

et al. 1993

Antimicrob Agents Chemother

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Plasmid pJSW101 derived from pUC19 and carrying the wild-type gyrA gene was found to be unstable in HM72, a quinolone-resistant (QR) clinical isolate of Escherichia coli, and resulted in no change in quinolone MICs. MICs determined in the presence of ampicillin to ensure plasmid presence, however, resulted in complementation. HM72 was proved to have a gyrA mutation based on the DNA sequence of a 418-bp fragment of gyrA. DNA sequencing identified a common mutation encoding nolone resistance. These have included cloning of the gyrA and gyrB genes from resistant strains and identification of changes in the nucleotide sequence and purification of the GyrA and GyrB proteins to demonstrate drug resistance in enzyme activity conferred by one subunit. Because they are time-consuming, both of these methods are not readily suitable for testing more than a few clinical isolates. Certain common gyrA mutations also abolish a Hinfl restriction enzyme site and may be detected as a restriction fragment length polymorphism (4). A simpler popular method involves transformation of a resistant strain with a plasmid containing the wild-type gyrA' gene. Because of the dominance of ,gyrA+ over resistant gyrA alleles, such merodiploid transformed strains become more quinolone susceptible. When there is a decrease in resistance with this test, it is inferred that the original resistant strain contained a gyrA mutation.In this paper, we report studies with a uropathogenic clinical isolate in which the plasmid test was falsely negative, as shown by subsequent cloning and sequencing of its gyrA gene. We suggest a simple method for overcoming instability of the transformed gyrA plasmid. In addition, we report that the quinolone resistance conferred by another mutant locus, nfxD, which is dependent on the expression of resistant gyrA, is also abolished by transformation with a plasmid containing gyrA . Thus, inferences drawn from the plasmid test about the level of resistance attributable to a gyrA mutation alone may also be subject to misinterpretation. MATERIALS AND METHODSBacterial strains (Table 1). E. coli HM72 was isolated from the urine specimen of an 81-year-old patient during treatment with pipemidic acid; this strain was resistant to nali-

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Rapid Method for Screening Large Numbers of Escherichia coli Colonies for Production of Plasmid-Mediated β-Lactamases

Abstract: A rapid, simple assay for screening large numbers of Escherichia coli colonies for production of certain plasmid-mediated P-lactamases (including TEM-1, TEM-2, HMS-1, SHV-1, OXA-1, PSE-1, PSE-4, and CEP-2) is described.

Cited by 11 publications

References 27 publications

Elimination of plasmid pMG110 from Escherichia coli by novobiocin and other inhibitors of DNA gyrase

Elimination of plasmid pMG110 from Escherichia coli by novobiocin and other inhibitors of DNA gyrase

Expression of the AsbA1, OXA-12, and AsbM1 beta-lactamases in Aeromonas jandaei AER 14 is coordinated by a two-component regulon

Limitations of plasmid complementation test for determination of quinolone resistance due to changes in the gyrase A protein and identification of conditional quinolone resistance locus

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