Purification and Properties of Dihydrostreptomyein-Phosphorylating Enzyme from Pseudomonas aeruginosa

The distribution of the dihydrostreptomycin (DHSM)-phosphorylating enzyme was investigated using DHSM-resistant strains of Pseudomonas aeruginosa, indicating that this enzyme was demonstrated from all of 7 DHSM-resistant strains examined but not from a DHSM-sensitive one. The DHSM-phosphorylating enzyme was isolated from P. aeruginosa TI-13 and purified about 205-fold using Sephadex G-75 and DEAE-Sephadex A-50 column chromatography. The optimal pH for the DHSM-inactivation was around 10.0, and both adenosinetriphosphate (ATP) and Mg⁺⁺ were required for the inactivating reaction. It was found that this enzyme inactivated only DHSM but not other aminoglycosidic antibiotics such as kanamycin, aminodeoxykanamycin, neomycin, paromomycin, lividomycin and gentamicin.     

Identification of Tn2401, a transposon encoding multiresistance to aminoglycosides

A transposable element, Tn2401, was found in a clinical isolate of Pseudomonas aeruginosa. Tn2401 had a size of 7190 nucleotides and encoded aminoglycoside 3'-phosphotransferase and aminoglycoside 6'-N-acetyltransferase. The sequence encoding the former enzyme was homologous with that of Tn903. Pseudomonas aeruginosa strains harbouring this transposon were resistant to kanamycin, neomycin, lividomycin, ribostamycin, paromomycin, netilmycin, tobramycin, dibekacin, gentamicin, sisomicin, and butirosin.     

Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics.

We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutants are sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramycin--antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS1, AGS2, and AGS3 (for aminoglycoside antibiotic sensitivity). Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants have intermediate antibiotic sensitivity. AGS1 was mapped 8 centimorgans distal from LEU2 on chromosome III. Analyses of yeast strains transformed with vectors carrying antibiotic resistance genes revealed that G418, gentamicin X2, kanamycin B, lividomycin A, neamine, and paromomycin are inactivated by the Tn903 phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strains are improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum of aminoglycoside antibiotics can be used to select for plasmid maintenance.     

Resistance to apramycin in Escherichia coli isolated from animals: detection of a novel aminoglycoside-modifying enzyme

The mechanisms of resistance to apramycin of five isolates of Escherichia coli from animals were investigated. Three isolates, which were resistant to all the aminoglycosides tested, did not transfer their resistance and did not produce aminoglycoside-modifying enzymes. The fourth isolate, which was resistant to apramycin, tobramycin, gentamicin, kanamycin and neomycin but not to amikacin, owed its resistance to production of the acetyltransferase AAC(3)IV. The gene specifying this enzyme was carried on a transposon, Tn800, on a plasmid designated R1535. The fifth isolate was resistant to apramycin, neomycin and kanamycin but not to gentamicin, tobramycin or amikacin. It produced an acetyltransferase that readily acetylated only apramycin, neomycin and paromomycin, a compound that is closely related to neomycin. Synthesis of this enzyme was specified by a chromosomal gene located near pyrD at about 20 min on the map of the E. coli K12 chromosome.     

R Factor-Mediated Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa

Conjugal transferability of drug resistance was examined, in eleven Pseudomonas aeruginosa strains which were isolated in Frankfurt. Four R factors were demonstrated from three strains using P. aeruginosa as recipients but they were nontransferable to Escherichia coli K12. Two R factors, i.e., R_ms146 and R_ms147, mediated resistances to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), kanamycin (KM), lividomycin (LV), gentamicin C complex (GM) and 3′,4′-dideoxykanamycin B (DKB). They mediated the formation of aminoglycoside-inactivating enzymes, i.e., SM phosphotransferase, SM adenylyltransferase, KM and LV phosphotransferase 1, and GM and DKB 6′-N-acetyltransferase. TC resistance conferred by these R factors was due to impermeability of the drug. P. aeruginosa Ps 142 carried two kinds of R factor in one cell, R_ms148 (SM) and R_ms149 (SM·SA·GM·CPC) (CPC, carbenicillin). R_ms148 (SM) was transferable at a high frequency of 10^–1 and mediated the formation of SM phosphotransferase. R_ms149 mediated the formation of drug-inactivating enzymes, i.e., GM 3-N-acetyltransferase and β-lactamase, but did not inactivate SM. SM resistance was probably due to impermeability of the drug.     

Resistance to gentamicin and related aminoglycosides in Staphylococcus aureus isolated in Brazil

Isolates of Staphylococcus aureus obtained from a Brazilian university hospital were characterized in relation to resistance to gentamicin and related aminoglycosides. Thirty-six isolates were susceptible to methicillin (MSSA) and 14 were resistant (MRSA). All isolates were sensitive to nucleic acid-binding compounds. All MRSA isolates and one MSSA isolate were demonstrated to be resistant to gentamicin and were coincidentally resistant to amikacin, kanamycin, neomycin and tobramycin. Among the gentamicin sensitive MSSA isolates, five isolates were found to be resistant only to kanamycin/neomycin. The resistance to gentamicin (and related aminoglycosides: kanamycin and tobramycin) must be due to AAC(6')-APH(2") activity. As these isolates also showed resistance to neomycin, they must carry an additional genetic element, probably the one responsible for APH(3')III activity, which accounts for the high level of resistance to kanamycin and to amikacin. The resistance to kanamycin/neomycin in the gentamicin sensitive isolates could not be attributed to the AAD(4')(4") activity because of the tobramycin sensitivity, and so could be ascribed to the APH(3')III activity. Curing and transfer experiments, as well as electrophoresis procedures, indicate that gentamicin resistance in Staph. aureus strains here studied has, characteristically, chromosomal localization.     

Ribosomal resistance of an istamycin producer, Streptomycestenjimariensis, to aminoglycoside antibiotics

$\text{Streptomyces}\text{tenjimariensis}$ SS-939 was resistant to its own aminoglycoside antibiotics, istamycins, as well as kanamycin A, neamine, ribostamycin and butirosin A, but was susceptible to neomycin B, lividomycin A and streptomycin. This resistance to these antibiotics was found to be due to ribosomes of the strain.     

Drug resistance in Salmonellae isolated at Chandigarh (India) during 1972–1978

A total of 1316 strains of Salmonella belonging to 20 serotypes isolated at P.G.I. Chandigarh (India) were tested for drug resistance. Drug resistance was noticed in 494 (38.3%) of the strains; 194 (14.8%) of these strains were resistant to one drug, while 300 (23.5%) had multiple drug resistance. All isolated strains were sensitive to gentamicin, furazolidone and nalidixic acid.Resistance to streptomycin was observed in 233 (17.7%), chloramphenicol 197 (14,9%), tetracycline 293 (22.3%), ampicillin 428 (32.5%), kanamycin 206 (15.7%), neomycin 206 (15.7%) and sulphadiazine 215 (19.9%).Multiple drug resistance was most common in S. bareilly, S. typhimurium and S. anatum serotypes. Increase in incidence of drug resistance in Salmonellae has been noticed during 1972–1978.     

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

Naturally occurring drug resistance plasmids in hospital strains of Staphylococcus aureus

A genetic analysis of multiply inorganic salts and antibiotic--resistant strains of Staphylococcus aureus was performed. Experiments designed to show reversion of organisms to antibiotic and inorganic salt susceptibility, as well as studies on the influence of ultraviolet irradiation of phage on the transduction frequencies of the resistance markers, indicated that determinants of chloramphenicol, tetracycline, aminoglycoside antibiotics, inorganic salts, and penicillin resistance in hospital strain are present on separate plasmids. Transduced by us plasmids pN742 and pN794 determined resistance to neomycin, kanamycin, paromomycin, lividomycin and streptomycin.     

Inactivation of Kanamycin,Neomycin, and Streptomycin by Enzymes Obtained in Cells of Pseudomonas aeruginosa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 × g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3′-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.     

Aminoglycosides resistance in clinical isolates of Staphylococcus aureus from a University Hospital in Bialystok, Poland

Staphylococcus aureus obtained from a University Hospital in Poland were characterized in relation to resistance to aminoglycoside antibiotics and the distribution of the genes encoding the most clinically relevant aminoglycoside modifying enzymes (AMEs). Of a total of 118 S. aureus, 45 (38.1%) isolates were found to be resistant to at least one of the tested antibiotics. All aminoglycoside resistant isolates except one 44 (97.8%) were resistant to kanamycin. The majority of strains 37 (82.2%) and 32 (71.1%) expressed resistance to neomycin and tobramycin, respectively. Eleven strains (24.4%) were resistant to gentamicin or amikacin. All S. aureus strains were sensitive to netilmicin. The most prevalent resistance gene was aac(6')-Ie+aph(2') found in 13 (28.9%) strains and 12 (26.7%) isolates carried ant(4')-Ia gene, whilst aph(3')-IIIa gene was detected in only 7 (15.6%) isolates. Additionally, the ant(6)-Ia and str genes were detected in 14 (31.1%) and 2 (4.4%) strains, respectively. Ten (22.2%) strains resistant to amikacin, tobramycin, kanamycin or neomycin did not harbor any of the above-noted genes.     

Frequency of R Factor-mediated Multiple Drug Resistance in Klebsiella and Aerobacter

A comparative study was done on the transfer frequency of R factors from 90 strains of multiple drug-resistant Aerobacter and 81 strains of Klebsiella to Escherichia coli CSH-2 (F(-), met(-), pro(-), Nal-r). The most common resistance patterns for the Aerobacter isolants were ampicillin streptomycin chloramphenicol tetracycline and ampicillin streptomycin chloramphenicol tetracycline kanamycin neomycin; for the Klebsiella isolants, the most common resistance pattern was ampicillin kanamycin streptomycin tetracycline chloramphenicol neomycin. R factors were isolated from 14.1% of the Aerobacter strains; 61.5% of these R factors harbored R determinants for ampicillin streptomycin tetracycline. R factors were isolated from 79.1% of the Klebsiella strains; four R factors were isolated with significant frequency; streptomycin chloramphenicol kanamycin neomycin, 37.5%; ampicillin streptomycin tetracycline kanamycin neomycin, 14.1%; ampicillin streptomycin tetracycline, 12.5%; and streptomycin chloramphenicol tetracycline, 12.5%.Chloramphenicol, kanamycin, and neomycin resistance was rarely transferred from the Aerobacter strains, although over 50% of the clinical isolants possessed resistance to these antibiotics. In contrast, over 75% of the Klebsiella strains transferred resistance to chloramphenicol, kanamycin, neomycin. Highest frequency of transferred resistance to individual drugs in the Aerobacter strains was to streptomycin (14.8%), whereas in the Klebsiella group resistance to four drugs was transferred at a very high frequency: streptomycin (80.8%), chloramphenicol (78.5%), kanamycin (76.4%), and neomycin (75.9%).     

Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation.

The binding of [${}^3\text{H}$]kanamycin to $\text{E. coli}$ ribosomes and ribosomal subunits was studied by equilibrium dialysis and Millipore filter methods. The 70S ribosome bound ca. two molecules up to the antibiotic concentration of 10 uM, and more at higher concentrations. Each ribosomal subunit was observed to possess one major binding site, and the affinity of the small ribosomal subunit was greater than that of the large subunit. The binding of [${}^3\text{H}$]kanamycin to ribosomes and ribosomal subunits was reversed by neomycin or gentamicin, but not by streptomycin and chloramphenicol. Kanamycin, neomycin and gentamicin interfered with the binding of [${}^{14}\text{C}$] tuberactinomycin O. Translocation of N-Ac-Phe-tRNA was markedly inhibited by kanamycin, neomycin or gentamicin, but not by streptomycin.     

The role of aminoglycoside--modifying enzymes in resistance of Gram-negative rods

The aim of the study was to evaluate the aminoglycoside resistance of Gram-negative bacilli isolated from patients. To the examination 35 strains of Enterobacteriaceae and 18 of non-fermentative bacteria were included. Resistance to aminoglycosides (gentamicin (G), netilmicin (Nt), tobramycin (T), amikacin (A), kanamycin (K), neomycin (N)) was established by disk diffusion method. Interpretation of enzymatic mechanisms was performed by Livermore. The most common enzymes AAC(6')I were found in Enterobacteriaceae group (mostly in E. cloaceae and P. mirabilis) and AAC(3') and in non-fermentative bacteria: AAC(6')I in P. aeruginosa and APH(3')VI and AAC(3')I in A. baumanii. The most frequent phenotype was resistance to six antibiotics (G, Nt, T, A, K, N) Resistance rates were high for gentamicin (>70 %) in both groups and amikacin (88,89 %) in non-fermentatives.     

分离自母婴肠道的假小链双歧杆菌的耐药性分析

【背景】由于滥用抗生素导致细菌耐药性日益严重。对于双歧杆菌,人们往往注重其益生功能的挖掘而忽视了对其耐药性的研究,存在一定的安全隐患。【目的】检测母婴肠道中假小链双歧杆菌的耐药性,探究婴儿肠道中假小链双歧杆菌耐药性的来源。【方法】利用微量肉汤稀释法测定48株分离自母婴肠道的假小链双歧杆菌对14种抗生素的耐药性,比较分离自不同家庭母婴肠道中假小链双歧杆菌的耐药性。【结果】48株母婴肠道分离株对四环素、氯霉素、新霉素、环丙沙星100%耐药,对其余10种抗生素耐药率依次为：卡那霉素98%、利福平80%、克林霉素78%、甲氧苄啶63%、红霉素59%、庆大霉素43%、链霉素16%、万古霉素14%、氨苄西林6%、利奈唑胺2%。母婴肠道分离株的耐药性无显著差异,分离自同一家庭母婴肠道的菌株具有相似的耐药表型。【结论】分离自母婴肠道的假小链双歧杆菌对多种抗生素具有耐药性,婴儿肠道中假小链双歧杆菌的耐药性可能是由母亲肠道垂直传递而来。     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.     

New Plasmid-Mediated Phosphorylation of Gentamicin C in Staphylococcus aureus

A clinical isolate of Staphylococcus aureus resistant to gentamicin, kanamycin and 3′,4′-dideoxykanamycin B contained two enzymes capable of inactivating gentamicin, i.e., an aminoglycoside 2″-phosphotransferase and aminoglycoside acetyltransferase.     

Transfer of plasmid-borne resistance from a multiply-resistant Staphylococcus aureus isolate,WBG1022

Staphylococcus aureus isolate, WBG1022, was resistant to penicillin, kanamycin, neomycin, streptomycin, chloramphenicol, trimethoprim, cadmium, and ethidium bromide and harbored plasmids of 34.5, 24.5, 4.4, 3.2, and 2.6 kilobases. The plasmids were transferred in mixed-culture transfer and conjugation experiments. No resistance phenotype was associated with the 2.6-kb plasmid. The 3.2-kb and 4.4-kb plasmids encoded chloramphenicol and streptomycin resistance respectively. The 24.5-kb plasmid, pWBG626, encoded joint resistance to penicillin, kanamycin, neomycin, and ethidium bromide. Resistance to trimethoprim and cadmium were chromosomal. The 34.5-kb plasmid, pWBG661, had no resistance phenotype but was found to be conjugative. It also mobilized the 4.4-kb and 24.5-kb plasmids in WBG1022. Restriction endonuclease analysis of pWBG661 with EcoRI, ClaI, PvuII, and BglII restriction enzymes demonstrated that pWBG661 was identical to two previously isolated S. aureus conjugative plasmids, p WBG620 and pWBG637, that also lack resistance phenotypes.     

Inactivation of kanamycin, neomycin, and streptomycin by enzymes obtained in cells of Pseudomonas aeruginoa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 x g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3'-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.