KAYSER, F. H.

MAINO, L.

SANTANAM, P.

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Journal of Antimicrobial Chemotherapy (1985) 16,401-408CorrespondenceA unique aminoglycoside-O-phosphorylating transferred the kanamycin resistance alongactivity mediating resistance to aminoglycosides with ampicillin resistance to the recipient strainin Escherichia colt HK 254 (F ~, R ", Lac " ) by conjugation (TableI), at a frequency of 10 "2 and 10 " 3 respec-Sir, tively. However, strain PS 5 alone transferredThe limitation of disc diffusion tests was resistance to tetracycline as well. The transferrecently illustrated by a report on the failure of of resistance markers in E. coli PS 5 wasthe test to detect moderate levels of resistance mediated by a plasmid that was > 54-2 kb andto tobramycin in kanamycin-resistant strains that could be eliminated by treatment withof Escherichia coli (Santanam, 1984). The acriflavin. The resulting derivative wasresistance in two representative strains was phenotypically susceptible to kanamycin,found to be mediated by an amfnoglycoside- streptomycin, ampicillin and tetracycline byO-phosphorylating enzymatic activity having disc diffusion tests (Table I). Strain PS 10, ina novel spectrum of substrates. This report addition to a large plasmid similar to the onepresents the results of additional studies on the found in PS 5, had two other detectablephosphorylating activity and its genetic basis in plasmids with a size of 6-1 kb and 5-4 kb.the two strains. However, treatment of PS 10 with acriflavinThe two strains PS 5 and PS 10, having a selectively eliminated resistance only tomoderate level of resistance to gentamicin and kanamycin. The resulting derivative wastobramycin (MICs '5 mg/1), were susceptible to phenotypically susceptible only to kanamycin,amikacin (MIC 13 mg/1) and netilmicin (MIC but not to streptomycin, ampicillin or tetra-6 mg/1) (National Committee for Clinical cycline. It retained all the plasmids found in theLaboratory Standards, 1982). Both the strains wild strain, without any apparent change dueTable I. Resistance pattern of E. coli strains used in mating experiments'E. coli strainHK254(F",R-)Lac-PS 5 Lac+PS5-HK254PS5(E)PS 10 Lac+PS10-HK254PS 10 (E)RecipientDonorTrans-conjEliminanf1DonorTrans-conjEliminant>SRRRSRRRKSRRSRRS"Disc diffusion test resulAMSRRSRRRCFSSSsRSRtCssssRssTsRRSRSS°R, Resistant; S, susceptible*S, Streptomycin; K., kanamycin; AM, ampicillin; CF, cephalothin; C, chloramphenicol; T, tetracyclinecMating was performed first by growing for 4 h at 37°C cultures of the donor and the recipient grown over-night and diluted in brain-heart infusion (BHI) to 1:10 and 1:100 respectively. One millilitre of the donorculture was mixed with 4-5 ml of the recipient, the volume made up to 10 ml in BHI and incubated at 37°C.Transconjugants were selected from 100 ul samples taken after 2 h and 24 h of incubation, on Mackonkey agarincorporating 4000 mg/1 of streptomycin and 500 mg/1 of kanamycin.'Eliminants were selected from BHI cultures incorporating with 16 mg/1 of acriflavin stored at 37°C for 4weeks.401402 CorrespondenceTable II. Phosphorylation of aminoglycosides by the cell extract of E. coli PS 10DrugRibostamycinParomomycinNeomycinButirosinKanamycinLividomycinStreptomycinNetilmicinAmikacinGentamicinSisomicinTobramycinDibekacinSpectinomycin32P incorporated°(cpm/3 nmol)46,15438,82735,81122,04121,42316,42811,7978740810677227519294029410Efficiency(%)100841277-5947-7646-4235-5925-5618-9417-5616-7316-296-376-370*MIC(mg/1)>100>100>100>100>100>100>1006135—5——Totalinactivation3 nmoles3 nmoles3 nmoles3 nmoles3 nmoles3 nmoles3 nmoles80 pmoles80 pmoles80 pmoles80 pmoles80 pmoles80 pmolesnot tested° A value of 978 cpm from the control without drug has been deducted from the experimental values.* MIC was tested by spreading a suspension of 1 x 10' to 4 x 10' cells in 100 ul of Mueller-Hinton broth onMueller-Hinton agar containing an aminoglycoside as described elsewhere (Santanam, 1984).' Total inactivation was determined by the microbiological assay (Santanam & Kayser, 1976a).to loss of the kanamycin resistance as discernedby agarose gel electrophoresis of plasmid DNA(Kieser, 1984).The cell extract obtained by the osmoticshock procedure (Santanam & Kayser, 19766),phosphorylated ribostamycin with maximumefficiency (Table II). The efficiency of phos-phorylation of netilmicin and amikacin was16% that of ribostamycin and that of tobramy-cin and dibekacin was only 6%. Phosphoryla-tion resulted in the inactivation of the drugstested in the range of 3 nmoles to 80 pmoles(Santanam & Kayser, 1976a).The phosphorylating activity described herehas a unique spectrum of substrates thatencompasses the spectrum of substrates of bothaminoglycoside-O-phosphotransferase (3') IIIand aminoglycoside-O-phosphotransferase(2") (Foster, 1983) both of which are usuallyassociated with Gram-positive organisms.Since 3', 4'-dideoxykanamycin B (dibekacin)and gentamicin, in addition to tobramycin,are substrates for the enzymatic activity,phosphorylation occurs presumably in thedeoxystreptamine moiety (ring II) or amino-hexose HI of the aminoglycoside molecule.Whether phosphorylation of the differenthydroxyl groups is mediated by a single novelenzyme or by a combination of enzymesremains to be determined, as does the exactmolecular weight of the large plasmid.This work was presented in part at the 4thMediterranean Congress of Chemotherapyheld in Rhodos, Greece, during October 1984(abstract no. 404).P. SANTANAML. MAINOF. H. KAYSERInstitute for Medical Microbiology,University of Zurich.Gloriastrasse-32,. CH-8028 Zurich,SwitzerlandReferencesFoster, T. J. (1983). Plasmid-determined resistance toantimicrobial drugs and toxic metal ions in bac-teria. Microbiological Reviews 47,361-409.Kieser, T. (1984). Factors affecting the isolation ofccc DNA from Streptomyces lividans andEscherichia coli. Plasmid 12, 19-36.National Committee for Clinical Laboratory Stan-dards. (1982). Performance standards for anti-microbic disc susceptibility tests. Second Supple-ment, Vol. 2. National Committee for ClinicalLaboratory Standards, Villanova, Pa.Santanam, P. (1984). Failure of the disk diffusion testto detect tobramycin resistance in kanamycin-resistant Escherichia coli strains. Journal of ClinicalMicrobiology 20,295-7.Santanam, P. & Kayser, F. H. (1976a). Tobramycinadenylyltransferase: A new aminoglycoside inacti-vating enzyme from Staphylococcus epidermidis.Journal of Infectious Diseases 134, Suppl., S33-9.Santanam, P. & Kayser, F. H. (1976i). Enzymaticadenylylation by aminoglycoside 4'-adenylyl-transferase and 2"-adenylyltransferase as a meansof determining concentration of aminoglycosideantibiotics in serum. Antimicrobial Agents andChemotherapy 10,664-7.

A unique aminoglycoside-O-phosphorylating activity mediating resistance to aminoglycosides in Escherichia coli

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