Semisynthetic aminoglycoside antibiotics: Development and enzymatic modifications

The critical resistance mechanisms of aminoglycoside antibiotics in bacteria of clinical importance are the enzymatic N-acetylation, O-phosphorylation, and O-nucleotidylation that generally result in the inactivation of aminoglycosides. To overcome such resistance mechanisms, dibekacin (3′,4′-dideoxykanamycin B) was developed as the first rationally designed semisynthetic aminoglycoside, based on the enzymatic 3′-O-phosphorylation of kanamycin. Subsequently, amikacin, netilmicin, and isepamicin were developed by introducing (S)-4-amino-2-hydroxybutyryl (AHB), ethyl, and (S)-3-amino-2-hydroxypropionyl side chains into the 1-amino group of kanamycin, sisomicin, and gentamicin B, respectively. These side chains are believed to block the access of a variety of aminoglycoside-modifying enzymes to their target sites. The latest semisynthetic aminoglycoside of clinical use in Japan is arbekacin (1-N-AHB-dibekacin), which has been extensively used since its approval as an anti-methicillin-resistant Staphylococcus aureus (MRSA) agent in 1990. Although it has several possible modification sites for aminoglycoside acetyltransferases (AACs), arbekacin-resistant MRSA strains that have emerged in the past 8 years have been those with a low or moderate level of resistance, due to a bifunctional enzyme, AAC(6′)/APH(2″), at low incidence. To overcome AAC(6′)/APH(2″)-dependent arbekacin-resistant MRSA strains, 2″-amino-2″-deoxyarbekacin and its 5-epiamino derivative have been already synthesized. However, simulative modification studies using AACs from aminoglycoside-producing Streptomyces strains have revealed that AAC(3) and AAC(2′) converted arbekacin to 3″-N-acetyl and 2′-N-acetyl derivatives, respectively, which retain high antibiotic activity. By contrast, the same acetylations of amikacin (3″-N-) and dibekacin (3-N-) resulted in their inactivation. Thus, these new findings confirmed the steric hindrance effect of the 1-N-acyl side chain and illuminated the novel aspect of arbekacin distinct from the other semisynthetic aminoglycosides, indicating that MRSA strains cannot be arbekacin-resistant even if they have acquired the aac(3) or aac(2′) gene. Received: November 26, 1998 / Accepted: December 9, 1998     

Emergence of 4'',4"-aminoglycoside nucleotidyltransferase in enterococci.

Enterococcus faecium BM4102 was resistant to macrolide-lincosamide-streptogramin B-type (MLS) antibiotics; tetracycline-minocycline; and high levels of kanamycin, neomycin, tobramycin, and dibekacin but not gentamicin. This aminoglycoside resistance phenotype is new in enterococci. The genes conferring resistance to aminoglycosides and MLS antibiotics in this strain were carried on a plasmid, pIP810, that was self-transferable to to other Enterococcus strains. Resistance to tobramycin and structurally related aminoglycosides, kanamycin, neomycin, and dibekacin, was due to synthesis of a 4',4"-aminoglycoside nucleotidyltransferase. Homology was detected by hybridization between pIP810 DNA and a probe specific for a gene encoding an enzyme with identical site specificity in staphylococci. The bacteriostatic activity of amikacin apparently was not affected by the presence of the enzyme, although it was modified in vitro. However, the bactericidal activity of amikacin and the synergism of this aminoglycoside with penicillin were abolished.     

In Vitro Comparison of Kanamycin, Kanendomycin, Gentamicin, Amikacin, Sisomicin, and Dibekacin Against 200 Strains of Pseudomonas aeruginosa

The antimicrobial activity of kanamycin, kanendomycin, gentamicin, amikacin, sisomicin, and dibekacin against 200 strains of Pseudomonas aeruginosa was compared. Dibekacin was found to be the most active against the tested organisms, whereas the other aminoglycoside antibiotics fell in the following order of diminishing antibacterial potency: amikacin, sisomicin, gentamicin, kanendomycin, and kanamycin. Seven strains showed high-level resistance to gentamicin (minimal inhibitory concentration, 400 mug/ml), and two of them were also resistant to amikacin and sisomicin (minimal inhibitory concentration, 75 mug/ml). The minimal inhibitory concentration of dibekacin for these seven strains was 0.625 mug/ml.     

Comparison of aminoglycoside resistance patterns in Japan, Formosa, and Korea, Chile, and the United States.

The resistance mechanisms of more than 2,000 aminoglycoside-resistant gram-negative aerobic bacteria were estimated by a method that assigned a biochemical mechanism based on susceptibility to selected aminoglycosides. Strains from hospitals in Japan, Formosa, and Korea (the Far East) were compared with strains from Chile and the United States. Of the strains from Chile, 90% had an aminoglycoside resistance pattern indicative of the 3-N-acetyltransferase [AAC(3)-V] enzyme. Of the strains from the Far East, 78% had susceptibility patterns suggesting the presence of AAC(6') enzymes. In contrast, strains from the United States had a wider variety of resistance mechanisms including 2'-O-adenylyltidyltransferase [ANT(2')], AAC(3), AAC(6'), and AAC(2'). Reflecting these differences in resistance patterns, the frequencies of resistance to gentamicin, tobramycin, dibekacin, and amikacin in strains from the United States were different from those in strains from the Far East. These differences seem to be correlated with different aminoglycoside usage in the two regions. In the United States, where gentamicin was the most widely used aminoglycoside, 92% of the strains were resistant to gentamicin, 81% were resistant to dibekacin, and 8.8% were resistant to amikacin. In the Far East, dibekacin and kanamycin were widely used in the past and more recently amikacin has been frequently used. Of the strains from this region, 99% were resistant to dibekacin, 85% were resistant to gentamicin, and 35% were resistant to amikacin.     

R Factor-Mediated Aminoglycoside Antibiotic Resistance in Pseudomonas aeruginosa: a New Aminoglycoside 6′-N-Acetyltransferase

The newly introduced semisynthetic aminoglycoside antibiotics, i.e., 3′,4′-dideoxykanamycin B (DKB), 6′-N-methyl DKB (6′-Me-DKB) and amikacin (AK) have been found to be effective against gram-negative pathogens including Pseudomonas aeruginosa, which are resistant to the known aminoglycoside antibiotics. We have demonstrated in our stock cultures two types of P. aeruginosa strains resistant to DKB, i.e., (DKB^r.AK^r.6′-Me-DKB^s) and (DKB^r.AK^s.6′-Me-DKB^r) (where r = resistant; s = sensitive). Both groups of strains inactivate the drugs by acetylation. The acetylating enzyme was extracted from GN4925(DKB^r.AK^s.6′-Me-DKB^r) and purified by affinity chromatography. Enzymatic studies of the inactivation reaction and chemical studies of the inactivated products indicated that DKB and 6′-Me-DKB were inactivated by acetylation of the 6′-amino group of the drugs. This enzyme acetylates kanamycin A (KM-A), KM-B, DKB, 6′-Me-DKB, 6′-N-methyl kanamycin B, but not KM-C, AK, and gentamicin C₁. The enzyme is named aminoglycoside 6′-N-acetyltransferase 3. Genetic studies of two strains resistant to DKB and 6′-Me-DKB disclosed that the enzyme catalyzing inactivation of both DKB and 6′-Me-DKB was mediated by an R factor, i.e., R_ms167 and R_ms168, capable of conferring resistance to KM, DKB, and 6′-Me-DKB, in addition to resistance to gentamicin, streptomycin, and sulfanilamide, and resistance to tetracycline, chloramphenicol, streptomycin and sulfanilamide respectively.     

Cosubstrate Tolerance of the Aminoglycoside Resistance Enzyme Eis from Mycobacterium tuberculosis

We previously demonstrated that aminoglycoside acetyltransferases (AACs) display expanded cosubstrate promiscuity. The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis is responsible for the resistance of this pathogen to kanamycin A in a large fraction of clinical isolates. Recently, we discovered that Eis is a unique AAC capable of acetylating multiple amine groups on a large pool of aminoglycoside (AG) antibiotics, an unprecedented property among AAC enzymes. Here, we report a detailed study of the acyl-coenzyme A (CoA) cosubstrate profile of Eis. We show that, in contrast to other AACs, Eis efficiently uses only 3 out of 15 tested acyl-CoA derivatives to modify a variety of AGs. We establish that for almost all acyl-CoAs, the number of sites acylated by Eis is smaller than the number of sites acetylated. We demonstrate that the order of n-propionylation of the AG neamine by Eis is the same as the order of its acetylation. We also show that the 6′ position is the first to be n-propionylated on amikacin and netilmicin. By sequential acylation reactions, we show that AGs can be acetylated after the maximum possible n-propionylation of their scaffolds by Eis. The information reported herein will advance our understanding of the multiacetylation mechanism of inactivation of AGs by Eis, which is responsible for M. tuberculosis resistance to some AGs.     

Aminoglycoside resistance resulting from tight drug binding to an altered aminoglycoside acetyltransferase

The aacA29b gene, which confers an atypical aminoglycoside resistance pattern to Escherichia coli, was identified on a class 1 integron from a multidrug-resistant isolate of Pseudomonas aeruginosa. On the basis of amino acid sequence homology, it was proposed that the gene encoded a 6'-N-acetyltransferase. The resistance gene was cloned into the pET23a(+) vector, and overexpression conferred high-level resistance to the usual substrates of the aminoglycoside N-acetyltransferase AAC(6')-I, except netilmicin. The level of resistance conferred by aacA29b correlated perfectly with the level of expression of the gene. The corresponding C-terminal six-His-tagged AAC(6')-29b protein was purified and found to exist as a dimer in solution. With a spectrophotometric assay, an extremely feeble AAC activity was detected with acetyl coenzyme A (acetyl-CoA) as an acetyl donor. Fluorescence titrations of the protein with aminoglycosides demonstrated the very tight binding of tobramycin, dibekacin, kanamycin A, sisomicin (K(d), 相似文献   

Identification of a Novel 6′-N-Aminoglycoside Acetyltransferase,AAC(6′)-Iak,from a Multidrug-Resistant Clinical Isolate of Stenotrophomonas maltophilia

Stenotrophomonas maltophilia IOMTU250 has a novel 6′-N-aminoglycoside acetyltransferase-encoding gene, aac(6′)-Iak. The encoded protein, AAC(6′)-Iak, consists of 153 amino acids and has 86.3% identity to AAC(6′)-Iz. Escherichia coli transformed with a plasmid containing aac(6′)-Iak exhibited decreased susceptibility to arbekacin, dibekacin, neomycin, netilmicin, sisomicin, and tobramycin. Thin-layer chromatography showed that AAC(6′)-Iak acetylated amikacin, arbekacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, and tobramycin but not apramycin, gentamicin, or lividomycin.     

In Vivo Antibacterial Activity of Vertilmicin,a New Aminoglycoside Antibiotic

Vertilmicin is a novel aminoglycoside antibiotic with potent activity against gram-negative and -positive bacteria in vitro. In this study, we further evaluated the efficacy of vertilmicin in vivo in systemic and local infection animal models. We demonstrated that vertilmicin had relatively high and broad-spectrum activities against mouse systemic infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. The 50% effective doses of subcutaneously administered vertilmicin were 0.63 to 0.82 mg/kg, 0.18 to 0.29 mg/kg, 0.25 to 0.99 mg/kg, and 4.35 to 7.11 mg/kg against E. coli, K. pneumoniae, S. aureus, and E. faecalis infections, respectively. The therapeutic efficacy of vertilmicin was generally similar to that of netimicin, better than that of gentamicin in all the isolates tested, and better than that of verdamicin against E. coli 9612 and E. faecalis HH22 infections. The therapeutic efficacy of vertilmicin was further confirmed in local infection models of rabbit skin burn infection and mouse ascending urinary tract infection.Aminoglycosides are a group of highly potent, broad-spectrum bactericidal antibiotics (8). Their history began with the discovery of streptomycin (12), followed by kanamycin, gentamicin, tobramycin, and a series of semisynthetic aminoglycosides (dibekacin, amikacin, and netilmicin) for the treatment of resistant organisms (8). The mechanisms of aminoglycoside resistance involved (i) modifying enzymes (the most common mechanism), (ii) mutations of the ribosomal binding site (causes resistance to streptomycin), and (iii) reduced drug uptake (mostly seen in Pseudomonas spp.) (2, 13). The semisynthetic aminoglycosides are mainly designed for the treatment of organisms that have developed resistance by producing aminoglycoside-modifying enzymes, i.e., N-acetyltransferase, O-nucleotidyltransferase, and O-phosphotransferases (8).Vertilmicin is a novel semisynthetic aminoglycoside derived from verdamicin. Our earlier study showed that it had broad in vitro antimicrobial activity which is similar to that of netilmicin and has the advantage of lower susceptibility to N-acetyltransferase 6′-Ie modification (5). In this study, we further investigated the in vivo antibacterial activities of this agent in a systemic infection model, as well as local infection models, to fill the gap between in vitro characterization and clinical evaluation. All of our animals studies were approved by the Animal Research Committee of the Institute of Medicinal Biotechnology.     

Transferrable Resistance to Tobramycin in Klebsiella pneumoniae and Enterobacter cloacae Associated with Enzymatic Acetylation of Tobramycin

Among gram-negative bacilli isolated from burn wound cultures, some strains of Enterobacteriaceae were resistant to tobramycin (minimal inhibitory concentration [MIC]≥ 20 μg/ml) but susceptible to gentamicin (MIC ≤ 5 μg/ml). One Klebsiella pneumoniae and two Enterobacter cloacae strains were selected for studies on their mechanisms of resistance to aminoglycoside antibiotics. Resistance to high concentrations of tobramycin (MICs of 25 to 50 μg/ml) was conjugally transferred to a susceptible Escherichia coli strain at rates of 1.2 × 10⁻⁴ to 2.8 to 10⁻⁴ per donor cell, suggesting that resistance is controlled by R factors. Resistances to tobramycin, kanamycin, and neomycin were cotransferred. Enzymatic activities were present that acetylated tobramycin, gentamicin, and kanamycin in osmotic lysates from the donor and transcipient strains. Enzymatic adenylylation of these aminoglycosides was not observed. The aminoglycoside-acetylating activities from K. pneumoniae and E. cloacae resembled kanamycin acetyltransferase (KAT) in their specificity for aminoglycoside substrates. Not all isolates of bacteria that produce KAT are resistant to tobramycin, but the factors that determine susceptibility or resistance to tobramycin in KAT-producing bacteria have not yet been established.     

Novel Aminoglycoside 2″-Phosphotransferase Identified in a Gram-Negative Pathogen

Aminoglycoside 2″-phosphotransferases are the major aminoglycoside-modifying enzymes in clinical isolates of enterococci and staphylococci. We describe a novel aminoglycoside 2″-phosphotransferase from the Gram-negative pathogen Campylobacter jejuni, which shares 78% amino acid sequence identity with the APH(2″)-Ia domain of the bifunctional aminoglycoside-modifying enzyme aminoglycoside (6′) acetyltransferase-Ie/aminoglycoside 2″-phosphotransferase-Ia or AAC(6′)-Ie/APH(2″)-Ia from Gram-positive cocci, which we called APH(2″)-If. This enzyme confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested. Steady-state kinetic studies demonstrated that GTP, and not ATP, is the preferred cosubstrate for APH(2″)-If. The enzyme phosphorylates the majority of 4,6-disubstituted aminoglycosides with high catalytic efficiencies (k_cat/K_m = 10⁵ to 10⁷ M⁻¹ s⁻¹), while the catalytic efficiencies against the 4,6-disubstituted antibiotics amikacin and isepamicin are 1 to 2 orders of magnitude lower, due mainly to the low apparent affinities of these substrates for the enzyme. Both 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates of APH(2″)-If, but are inhibitors. The antibiotic susceptibility and substrate profiles of APH(2″)-If are very similar to those of the APH(2″)-Ia phosphotransferase domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme.     

Substrate-labeled fluorescent immunoassay for measuring dibekacin concentrations in serum and plasma.

We have developed a homogeneous substrate-labeled fluorescent immunoassay for the measurement of dibekacin concentrations in serum and plasma. The fluorogenic enzyme substrate beta-galactosyl-umbelliferone was covalently attached to tobramycin, an aminoglycoside structurally similar to dibekacin, to prepare a fluorogenic drug reagent (FDR). The FDR is nonfluorescent under assay conditions, but fluoresces upon hydrolysis by beta-galactosidase. However, binding of the FDR by antiserum to the cross-reactive drug kanamycin prevents enzyme hydrolysis. The fixed level of FDR competes with dibekacin within the sample for the limiting number of antibody-binding sites in the reaction mixture. Unbound FDR is hydrolyzed by beta-galactosidase to release a fluorescent product that is proportional to the dibekacin concentration in the sample. The assay exhibits good precision, standard curve reproducibility, recovery, sensitivity, and correlation with a comparative method. Additionally, the substrate-labeled fluorescent immunoassay is rapid and easy to perform.     

The prevalence of aminoglycoside-modifying enzymes among coagulase negative staphylococci in Iranian pediatric patients

In spite of widespread emergence of aminoglycoside resistance, these drugs are still used in the treatment of staphylococcal infections. This study aimed to investigate the distribution of aminoglycoside resistance and genes encoding aminoglycoside – modifying enzymes (AMEs) as well as Staphylococcal Cassette Chromosome mec (SCCmec) type in coagulase negative staphylococci (CoNS) in pediatric patients. Totally, 93 CoNS isolates were examined for susceptibility to aminoglycosides using disk diffusion and/or E-test methods. AMEs genes and SCCmec types were detected using multiplex PCR. Strain typing was performed using repetitive extragenic palindromic (REP) – PCR assay. The non-susceptibility rates to kanamycin, tobramycin, gentamicin, amikacin and netilmicin were 73%, 59%, 49.5%, 16% and 7.5%, respectively. aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia and aph(3′)-IIIa were encountered in 56 (60.2%), 38 (40.8%) and 18 (19.3%) isolates, respectively. In aac(6′)-Ie-aph(2″)-Ia- positive isolates, the non- susceptibility rates to kanamycin, gentamicin, tobramycin, amikacin and netilmicin were 83%, 74%, 73%, 49% and 43%, respectively. SCCmec types included type IV (n = 31), I (n = 17), II (n = 5), III (n = 4), and V (n = 2). Three isolates had two types; I + III (n = 2) and III + IV (n = 1) whereas 11 isolates were non-typeable. AMEs genes carriers were distributed frequently into type IV. We found diverse fingerprint patterns among our isolates. In conclusion, there was a strong correlation between alarming rate of aminoglycoside resistance and methicillin resistance. Discordances between phenotypic and genotypic detection of aminoglycoside resistance were discernible. AMEs genes might be related to SCCmec types.     

Decreased susceptibility to 4''-deoxy-6''-N-methylamikacin (BB-K311) conferred by a mutant plasmid in Escherichia coli

Escherichia coli MP6 contains a plasmid that encodes aminoglycoside 3'-phosphotransferase II, which phosphorylates kanamycin and confers high-level kanamycin resistance, Amikacin is a minor substrate of this enzyme, but MP6 is susceptible to amikacin. Strain MP10 has a spontaneous mutation in the plasmid of MP6 that increases the aminoglycoside 3'-phosphotransferase II activity not only against kanamycin but also against amikacin. This mutation is also responsible for the appearance of resistance to amikacin in MP10. Resistance to 4'-deoxy-6'-N-methylamikacin (BB-K311) by enzymatic modification has not been reported previously. As with amikacin, MP6 was susceptible to BB-K311 and its aminoglycoside 3'-phosphotransferase II did not phosphorylate this amikacin derivative appreciably. We found that the plasmid-borne mutation in MP10, however, localized by being cloned with a 3.7-megadalton HindIII fragment containing the aminoglycoside 3'-phosphotransferase II gene, resulted in increased phosphorylation of BB-K311 and resistance to it. Thus, the mutation distinguishing MP6 and MP10 has increased the activity of an existing aminoglycoside-modifying enzyme and produced new bacterial resistance to two previously minor substrates of the enzyme.     

Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.

A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.     

In Vitro Synergism Between Carbenicillin and Aminoglycosidic Aminocyclitols Against Acinetobacter calcoaceticus var. anitratus

Acinetobacter caleoaceticus var. anitratus is a nonfermentative, gram-negative bacillus that has been demonstrated to cause severe infections, usually in hospitalized patients. Since mild to moderate resistance of A. calcoaceticus to one or more aminoglycosidic aminocyclitols has been noted to occur, a study was undertaken to evaluate the activity of combinations of carbenicillin with either kanamycin, tobramycin, or gentamicin against 28 isolates of A. calcoaceticus obtained from clinical sources. Synergism (defined as at least 100-fold-increased killing at 24 h by the combination as compared with the most efficacious of the individual antibiotics) was demonstrated against 26 of 28 strains of A. calcoaceticus with carbenicillin plus kanamycin and carbenicillin plus tobramycin and against 25 of 28 strains with carbenicillin plus gentamicin. The median increased killing for the 28 strains was 4.2 log₁₀ with carbenicillin plus kanamycin and with carbenicillin plus tobramycin and 3.1 log₁₀ with carbenicillin plus gentamicin. The most important determinant of synergistic potential of each combination was the level of resistance of each strain of A. calcoaceticus to the aminoglycoside component of the combination.     

Characterization of Class 1 integrons from Pseudomonas aeruginosa that contain the bla(VIM-2) carbapenem-hydrolyzing beta-lactamase gene and of two novel aminoglycoside resistance gene cassettes

Two clonally unrelated Pseudomonas aeruginosa clinical strains, RON-1 and RON-2, were isolated in 1997 and 1998 from patients hospitalized in a suburb of Paris, France. Both isolates expressed the class B carbapenem-hydrolyzing beta-lactamase VIM-2 previously identified in Marseilles in the French Riviera. In both isolates, the bla(VIM-2) cassette was part of a class 1 integron that also encoded aminoglycoside-modifying enzymes. In one case, two novel aminoglycoside resistance gene cassettes, aacA29a and aacA29b, were located at the 5' and 3' end of the bla(VIM-2) gene cassette, respectively. The aacA29a and aacA29b gene cassettes were fused upstream with a 101-bp part of the 5' end of the qacE cassette. The deduced amino acid sequence AAC(6')-29a protein shared 96% identity with AAC(6')-29b but only 34% identity with the aacA7-encoded AAC(6')-I1, the closest relative of the AAC(6')-I family enzymes. These aminoglycoside acetyltransferases had amino acid sequences much shorter (131 amino acids) than the other AAC(6')-I enzymes (144 to 153 amino acids). They conferred resistance to amikacin, isepamicin, kanamycin, and tobramycin but not to gentamicin, netilmicin, and sisomicin.     

Aminoglycoside-Inactivating Enzymes in Clinical Isolates of Streptococcus Faecalis: AN EXPLANATION FOR RESISTANCE TO ANTIBIOTIC SYNERGISM

Clinical isolates of enterococci (Streptococcus faecalis) with high-level resistance to both streptomycin and kanamycin (minimal inhibitory concentration >2,000 mug/ml), and resistant to synergism with penicillin and streptomycin or kanamycin were examined for aminoglycoside-inactivating enzymes. All of the 10 strains studied had streptomycin adenylyltransferase and neomycin phosphotransferase activities; the latter enzyme phosphorylated amikacin as well as its normal substrates, such as kanamycin. Substrate profiles of the neomycin phosphotransferase activity suggested that phosphorylation occurred at the 3'-hydroxyl position, i.e., aminoglycoside 3'-phosphotransferase. A transconjugant strain, which acquired high-level aminoglycoside resistance and resistance to antibiotic synergism after mating with a resistant clinical isolate, also acquired both enzyme activities. Quantitative phosphorylation of amikacin in vitro by a sonicate of the transconjugant strain inactivated the antibiotic, as measured by bioassay, and the phosphorylated drug failed to produce synergism when combined with penicillin against a strain sensitive to penicillin-amikacin synergism.No differences were found in the sensitivity of ribosomes from a sensitive and resistant strain when examined in vitro using polyuridylic acid directed [(14)C]-phenylalanine incorporation in the presence of streptomycin, kanamycin, or amikacin. Therefore, we conclude that aminoglycoside-inactivating enzymes are responsible for the aminoglycoside resistance, and resistance to antibiotic synergism observed in these strains.     

Multiple-Aminoglycoside-Resistant Mutants of Bacillus subtilis Deficient in Accumulation of Kanamycin

Three classes of spontaneous multiple-aminoglycoside-resistant (mar) mutants of Bacillus subtilis were isolated by plating on a low (1.2 μg/ml) concentration of kanamycin sulfate and were found to be resistant also to low concentrations of paromomycin, neomycin and gentamicin. The three classes could be distinguished one from another by their degree of cytochrome deficiency, respiration deficiency, and susceptibility to kanamycin lethality. A fluctuation test showed that the mutations were spontaneous and not induced by the conditions of selection. Representative strains from two classes of mutants (mar-2 and mar-3) accumulated aminoglycoside very poorly in comparison with the parent strain, whereas a strain of the third class (mar-1) inactivated aminoglycoside present in the growth medium. The mar-3 strain studied (aroD163) had previously been shown to be a menaquinone auxotroph (Farrand and Taber, 1973) and to be deficient in amino acid uptake (Bisschop et al., 1975). Such mutants, which are resistant to low concentrations of aminoglycosides, may be of use in elucidating the biochemical and genetic bases of certain bacterial transport systems.     

Rapid method for determination of kanamycin and dibekacin in serum by use of high-pressure liquid chromatography.

A rapid, simple, and accurate method for the determination of kanamycin and dibekacin in serum by use of high-pressure liquid chromatography is described. The serum proteins were precipitated with 3.5% perchloric acid containing sodium octanesulfonate. After centrifugation, a sample of the supernatant was directly injected into the chromatograph. The determination of kanamycin and dibekacin was performed by a combination of reverse-phase, ion-pair chromatography, postcolumn derivatization with o-phthalaldehyde, and fluorescence detection. The correlation coefficients with fluorescence polarization immunoassay were 0.996 for kanamycin and 0.957 for dibekacin.