Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in
            <i>Escherichia coli</i>
            Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

We isolated 11 nonconjugative plasmids mediating resistance to aminoglycoside antibiotics, including gentamicin, from Pseudomonas aeruginosa strains. Their genetic properties were investigated in both P. aeruginosa and Escherichia coli transformants. The plasmid molecular weights ranged from 11 x 106 to 24 x 106. A low level or complete absence of gentamicin resistance was observed when these plasmids were introduced into E. coli, but gentamicin resistance was restored when the plasmids were transferred back to P. aeruginosa from E. coli. Aminoglycoside-modifying enzyme activity was detected in P. aeruginosa harboring these plasmids, but was absent or greatly reduced in E. coli strains. This lack of expression may explain the observed decrease in aminoglycoside resistance.Plasmids isolated from Pseudomonas aeruginosa have been classified into 11 incompatibility groups. Most of the conjugative plasmids are nontransmissible to Escherichia coli strains, except for plasmids of the P-1 or P-3 group (12). Three reasons for this nontransferability to E. coli can be postulated: (i) nontransmissibility owing to some differences in cell surface structure; (ii) no replication of the plasmids after transmission to E. coli strains; and (iii) failure of resistance gene expression in the E. coli system. Some of the plasmids mobilized to E. coli by the presence of P-1 group plasmids can replicate by themselves in that host (12,13,16). In contrast, many plasmids are incapable of replication in E. coli, except by recombination with the mobilizing plasmid (13,20).Recently the isolation frequency of gentamicin-resistant P. aeruginosa strains has increased (3,4,14,18,21,24,26). We have also isolated plasmids mediating gentamicin resistance and transferable by conjugation to a recipient P. aeruginosa strain in 20% of the 91 gentamicinresistant P. aeruginosa isolates from clinical specimens in Japan. Gentamicin-resistance plasmids which were transfer deficient were also found in 56% of the gentamicin-resistant strains. In this report, we examined the genetic properties of such nonconjugative plasmids mediating gentamicin resistance in P. aeruginosa and also in E. coli after being introduced by transformation.MATERIALS AND METHODS Media. Heart infusion agar (Eiken Kagaku Co., Tokyo, Japan) and peptone water were used for the determination of drug resistance. Peptone water con-

Section: Resultsmentioning

confidence: 99%

Plasmid-mediated gentamicin resistance of Pseudomonas aeruginosa and its lack of expression in Escherichia coli

Kato¹,

Sato²,

Iyobe³

et al. 1982

“…These include negatively charged polar heads of cytoplasmic membrane phospholipids (9). After binding to polar phospholipids, Sm and Gm associate with membrane "transporters" on the basis of their positive charge but otherwise nonspecifically and are driven across the cytoplasmic membrane by the membrane potential (interior negative) (energy-dependent transport).…”

Section: Discussionmentioning

confidence: 99%

“…Uptake of DHS, amino acids, and glucose. Uptake of DHS and Gm was determined as previously described (5,6,9) with NB and CMM media. Uptake of glutamine, proline, spermidine, and arginine was performed on exponential-growth-phase cells grown in CMM.…”

Section: Methodsmentioning

confidence: 99%

Aminoglycoside-Resistant Mutation of Pseudomonas aeruginosa Defective in Cytochrome c ₅₅₂ and Nitrate Reductase

Bryan

Nicas

Holloway

et al. 1980

Self Cite

A gentamicin-resistant mutant of Pseudomonas aeruginosa PA0503 was selected after ethyl methane sulfonate mutagenesis. The strain, P. aeruginosa PA02401 had increased resistance to all aminoglycosides tested but exhibited no change for other antibiotics. The mutation designated aglA (aminoglycoside resistance) was 50% cotransducible with the 8-min ilvB,C marker on the P. aeruginosa chromosome. It showed a marked reduction in cytochrome c52 and nitrate reductase (Nar) and a change in terminal oxidase activity. Cytochrome c552 is a component of the P. aeruginosa Nar. No changes in succinate and reduced nicotinamide adenine dinucleotide dehydrogenases, ubiquinone content, Mg2"/Ca2" membrane adenosine triphosphatase, and energy coupling of electron transport to adenosine 5'-triphosphate synthesis were detected. Transport of gentamicin and dihydrostreptomycin was impaired in PA02401, but transport of proline, arginine, glutamine, glucose or the polyamine spermidine was not reduced. Ribosomes of PA02401, and PA0503 bound dihydrostreptomycin equally well, and cell extracts did not inactivate gentamicin or dihydrostreptomycin. Strain PA02401 is resistant to gentamicin and dihydrostreptomycin because of impaired transport of these compounds. The transport studies indicate a selective coupling of dihydrostreptomycin and gentamicin transport with terminal electron transport. This conclusion was supported by results from another mutant (PA0417-T2) with increased Nar activity, enhanced dihydrostreptomycin and gentamicin transport and a reduction in resistance to these drugs. These results are discussed in relation to a refined model for aminoglycoside transport and briefly relative to plasmid-mediated aminoglycoside resistance.

“…Enzymatic modification of aminoglycosides does not necessarily confer resistance to the drugs (1,3,4,10). We describe a clinical isolate, Serratia marcescens 75, which was highly susceptible to netilmicin and yet had a high level of aminoglycoside 2"-O-nucleotidyltransferase [ANT(2")] activity for netilmicin when assayed with excess aminoglycoside substrate.…”

mentioning

confidence: 99%

Correlation of aminoglycoside resistance with the KmS and Vmax/Km ratios of enzymatic modification of aminoglycosides by 2''-O-nucleotidyltransferase

DeHertogh¹,

Lerner²

1985

A clinical isolate, Serrafia marcescens 75, was found to be susceptible to netilmicin and yet had a high level of aminoglycoside 2"-O-nucleotidyltransferase activity for netilmicin. Kinetic studies of the partially purified enzyme revealed substrate inhibition for gentamicin and tobramycin at concentrations greater than 10-2 mM, but this was not observed for netilmicin. The MICs of the aminoglycosides tested exhibited a good inverse correlation with the K,. values for the enzyme and a direct correlation with the V,..1/K, ratios of the enzyme.Enzymatic modification of aminoglycosides does not necessarily confer resistance to the drugs (1, 3, 4, 10). We describe a clinical isolate, Serratia marcescens 75, which was highly susceptible to netilmicin and yet had a high level of aminoglycoside 2"-O-nucleotidyltransferase [ANT(2")] activity for netilmicin when assayed with excess aminoglycoside substrate. We examined the relationship between the MICs of netilmicin, tobramycin, and gentamicin and the kinetic parameters of the ANT(2') enzyme which modified each of these aminoglycosides.S. marcescens 75 was tested for susceptibility to aminoglycosides by an agar dilution method (7). Inocula of 104 CFU of cells grown overnight in brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.) were applied with a pronged replicator (7) onto Mueller-Hinton agar (BBL) containing the test antibiotics in twofold dilutions. The strain was highly resistant to gentamicin and tobramycin, but susceptible to netilmicin (Table 1).Sonic extracts of S. marcescens 75 were prepared for the assay of aminoglycoside-modifying activity as previously described (9). Aminoglycoside-modifying activity was assayed by adsorption of the radioactive product onto phosphocellulose filter disks (Whatman P-81) by the method of Haas and Dowding (5). For the adenylylating assay, the final concentrations of reagents were as follows: 0.033 M Tris-hydrochloride, pH 7.8; 7.5 mM MgCl2; 2.5 mM dithiothreitol; tobramycin and yet did not confer resistance to netilmicin, even in another genetic background, we transferred resistance to tobramycin into Escherichia coli JSRO-N, a plasmidfree, nalidixic acid-resistant recipient. Transconjugants were selected with a frequency of 5 x 10-6 per donor cell on Mueller-Hinton agar containing tobramycin at 12.5 ,ug/ml and nalidixic acid as the counterselecting agent at 25 ,ug/ml. The MICs of gentamicin, tobramycin, and netilmicin were 50, 50, and 1.56 ,ug/ml, respectively. Sonic extracts from two transconjugants chosen for study had adenylylating activity for gentamicin, tobramycin, netilmicin, and kanamycin at ca. 50% the level of the activity from the donor strain. Again, there was negligible acetylating or phosphorylating activity.An 18-liter culture of S. marcescens 75 grown in brain heart infusion broth containing 15 ,ug of gentamicin per ml was incubated with agitation at 37°C until the optical density at 600 nm was 1.5. The cells were harvested, washed twice with 0.15 M NaCl, and centrifuged again. The final pe...