Nucleic acid hybridisation with a probe specific for 3 aminoglycoside acetyltransferase type IV: A survey of resistance to apramycin and gentamicin in animal strains of Escherichia coli

Abstract Resistance to apramycin due to production of a 3-aminoglycoside acetyltransferase type IV (AAC(3)IV) has recently been detected among Gram-negative bacteria isolated in France from bovine clinical samples. 24 apramycin-resistant Escherichia coli strains isolated over the country, and epidemiologically unrelated, were studied by colony hybridization using an intragenic probe specific for AAC(3)IV. The results obtained indicated that the structural gene for the acetyltransferase was present in all the isolates tested and in the corresponding apramycin-resistant transconjugants. This observation demonstrates that resistance to apramycin by acetylation of the antibiotic has spread very rapidly in bovine Gram-negative bacteria.     

Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasmas in cell culture

The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[beta-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]- phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.     

Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique

A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3–5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (?I) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo‐denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

Oligopeptide uptake and aminoglycoside resistance in Escherichia coli K12

Previous reports have suggested that Escherichia coli K12 mutants defective in the expression of oligogopeptide permease protein A (OppA) exhibit reduced sensitivity to aminoglycosides due to altered permeability of the cell envelope. In this work, the role of the OppA protein, and the oligogopeptide permease (Opp) transport system has been evaluated, in the resistance to aminoglycosides using derivatives of the E. coli K12 SS320 strain selected for triornithine resistance or with a deletion of the complete opp operon. All tested mutants were defective in the uptake of tri- and tetra-peptides but did not expressed resistance to aminoglycosides. Additionally, complementation tests carried out with a plasmid encoding the OppA protein did not affect the sensitivity of the strains to these antibiotics. Taken together, these evidences indicate that the Opp uptake system, as well as the OppA protein, does not play a direct role in the sensitivity to aminoglycosides in E. coli K12.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Specific Antimicrobial Synergism of Synthetic Hydroxy Isothiocyanates with Aminoglycoside Antibiotics

Hydroxy isothiocyanates, especially 2-(4-hydroxy-phenyl)ethyl isothiocyanate (hITC), were examined for antimicrobial synergism with several antibiotics against Escherichia coli and Staphylococcus aureus, using a multiwell plate system. hITC had antibacterial synergism, specifically with aminoglycoside antibiotics. The synergism was observed in synthetic medium (M9 minimal medium) or soybean casein digest broth, but not in nutrient broth. Synergism was seen in the presence of certain sugars such as glucose, fructose, and maltose in the medium.     

Characterization of RbmD (Glycosyltransferase in Ribostamycin Gene Cluster) through neomycin production reconstituted from the engineered <Emphasis Type="Italic">Streptomyces fradiae</Emphasis> BS1

Amino acid homology analysis predicted that rbmD, a putative glycosyltransferase from Streptomyces ribosidificus ATCC 21294, has the highest homology with neoD in neomycin biosynthesis. S. fradiae BS1, in which the production of neomycin was abolished, was generated by disruption of the neoD gene in the neomycin producer S. fradiae. The restoration of neomycin by self complementation suggested that there was no polar effect in the mutant. In addition, S. fradiae BS6 was created with complementation by rbmD in S. fradiae BS1, and secondary metabolite analysis by ESI/MS, LC/MS and MS/MS showed the restoration of neomycin production in S. fradiae BS6. These gene inactivation and complementation studies suggested that, like neoD, rbmD functions as a 2-N-acetlyglucosaminyltransferase and demonstrated the potential for the generation of novel aminoglycoside antibiotics using glycosyltransferases in vivo.     

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.