Mechanistic insights into translation inhibition by aminoglycoside antibiotic arbekacin.

How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from ∼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by ∼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.

In vitro activity of arbekacin against multidrug-resistant gram-negative bacilli.

BACKGROUND: Arbekacin is a broad-spectrum aminoglycoside with activity against some Gram-positive and Gram-negative bacteria. METHODS: Arbekacin minimum inhibitory concentration (MIC) values were determined for 296 drug-resistant Gram-negative bacilli, and compared to previously determined plazomicin, amikacin, gentamicin, and tobramycin MIC values. RESULTS: The MIC values required to inhibit 50% and 90% of isolates (MIC50 and MIC90, respectively) were 16 and >128 µg/ml, respectively. CONCLUSIONS: Arbekacin showed similar MIC50 values to amikacin and gentamicin, a lower MIC50 value than tobramycin, and a higher MIC50 value than plazomicin.

In vitro and in vivo antimicrobial activity of TS2037, a novel aminoglycoside antibiotic.

To overcome serious methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa infections, we synthesized TS2037, 5,4″-diepi-arbekacin, a novel aminoglycoside antibiotic, and evaluated its biological properties. TS2037 showed broad-range, as well as robust antibacterial activities against Gram-positive and Gram-negative bacteria. The MIC50 and MIC90 of TS2037 against clinical isolates of MRSA (n = 54) were both 0.25 µg/mL, and no resistant strain was observed. The MIC50 and MIC90 of TS2037 against clinical isolates of P. aeruginosa (n = 54) were 1 and 4 µg/mL, respectively. TS2037 and arbekacin, anti-MRSA aminoglycoside, were more stable against AAC(6')-APH(2″), aminoglycoside-6'-N-acetyltransferase and 2″-O-phosphotransferase, produced by resistant S. aureus than gentamicin. Therapeutic efficacies of TS2037 in the mouse models of systemic infection with MRSA were superior to those of arbekacin, vancomycin, and linezolid. The efficacy of TS2037 against systemic infection caused by P. aeruginosa producing AAC(6')-II was superior to those of arbekacin and amikacin. In the nephrotoxicity risk screening, the release of free N-acetyl-ß-D-glucosaminidase from the kidney epithelial cell line after treatment with TS2037 at 2.5 and 5.0 µM were 2.0 and 2.1 (U/L), respectively, which were about two times higher than those of arbekacin. In conclusion, TS2037 exhibited the most potent antibacterial activity among aminoglycosides tested against both MRSA and P. aeruginosa in vitro and in vivo, although its nephrotoxicity risk remains to be improved.

Paraphaeosphaeride D and berkleasmin F, new circumventors of arbekacin resistance in MRSA, produced by Paraphaeosphaeria sp. TR-022.

Two new compounds, designated paraphaeosphaeride D (1) and berkleasmin F (2) together with a previously known compound, berkleasmin A (3), isolated from a culture broth of the fungus Paraphaeosphaeria sp. TR-022, proved to be new circumventors of arbekacin (ABK) resistance in methicillin-resistant Staphylococcus aureus (MRSA). The structures of 1 and 2 were elucidated by spectroscopic analyses, including various NMR experiments. All compounds showed 10-100 times ABK circumvention activities using the paper disc method and reduced the MIC values of ABK against MRSA from 16 µg ml(-1) to 4 µg ml(-1) (fourfold) using the agar dilution method. These new compounds might be promising lead compounds for developing circumventors of ABK resistance in MRSA.

Antimicrobial resistance of Staphylococcus aureus isolated from skin infections and its implications in various clinical conditions in Korea.

BACKGROUND: Periodic investigations into patterns of antimicrobial resistance can help to optimize the efficacy of treatment and limit the development of resistance. OBJECTIVES: The aim of this study was to update information on patterns of antimicrobial resistance in Staphylococcus aureus isolated from skin infections in South Korea. METHODS: We retrospectively analyzed clinical information and in vitro antimicrobial resistance data for 965 clinical S. aureus isolates obtained from skin infections during 2010-2013 in a university hospital in South Korea. RESULTS: The rate of resistance to oxacillin (methicillin-resistant S. aureus [MRSA]) was 47.4%. Similar rates of resistance to erythromycin (45.6%), fusidic acid (44.0%), and clindamycin (42.3%) were noted. The rate of resistance to mupirocin was 8.4%. Overall, 4.9% of isolates were resistant to both fusidic acid and mupirocin. None of the isolates showed resistance to habekacin, synercid, teicoplanin, or vancomycin. Generally, antimicrobial resistance rates did not increase from 2010 to 2013 except with reference to a few agents such as mupirocin and rifampin. Isolates from surgical patients, inpatients, non-dermatology outpatients, and adult patients showed relatively high rates of resistance to multiple antimicrobials. Resistance to mupirocin was not only lower than that to fusidic acid but was consistent across clinical contexts. CONCLUSIONS: The prevalence of MRSA in skin infections in South Korea did not increase during 2010-2013. Isolates from dermatology outpatients showed relatively lower rates of resistance to multiple antimicrobials than isolates from non-dermatology outpatients. Among topical antimicrobials, resistance to mupirocin was relatively low regardless of clinical condition.

AAC(3)-XI, a new aminoglycoside 3-N-acetyltransferase from Corynebacterium striatum.

Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI.

Synthesis and antibacterial activity of 4″ or 6″-alkanoylamino derivatives of arbekacin.

Arbekacin, an aminoglycoside antibiotic, is an important drug because it shows a potent efficacy against methicillin-resistant Staphylococcus aureus. However, resistance to arbekacin, which is caused mainly by the bifunctional aminoglycoside-modifying enzyme, has been observed, becoming a serious problem in medical practice. To create new arbekacin derivatives active against resistant bacteria, we modified the C-4″ and 6″ positions of its 3-aminosugar portion. Regioselective amination of the 6″-position gave 6″-amino-6″-deoxyarbekacin (1), and it was converted to a variety of 6″-N-alkanoyl derivatives (6a-z). Furthermore, regioselective modifications of the 4″-hydroxyl group were performed to give 4″-deoxy-4″-epiaminoarbekacin (2) and its 4″-N-alkanoyl derivatives (12 and 13). Their antibacterial activity against S. aureus, including arbekacin-resistant bacteria, was evaluated. It was observed that 6″-amino-6″-N-[(S)-4-amino-2-hydroxybutyryl]-6″-deoxyarbekacin (6o) showed excellent antibacterial activity, even better than arbekacin.

Arbekacin activity against contemporary clinical bacteria isolated from patients hospitalized with pneumonia.

Arbekacin is a broad-spectrum aminoglycoside licensed for systemic use in Japan and under clinical development as an inhalation solution in the United States. We evaluated the occurrence of organisms isolated from pneumonias in U.S. hospitalized patients (PHP), including ventilator-associated pneumonia (VAP), and the in vitro activity of arbekacin. Organism frequency was evaluated from a collection of 2,203 bacterial isolates (339 from VAP) consecutively collected from 25 medical centers in 2012 through the SENTRY Antimicrobial Surveillance Program. Arbekacin activity was tested against 904 isolates from PHP collected in 2012 from 62 U.S. medical centers and 303 multidrug-resistant (MDR) organisms collected worldwide in 2009 and 2010 from various infection types. Susceptibility to arbekacin and comparator agents was evaluated by the reference broth microdilution method. The four most common organisms from PHP were Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella spp., and Enterobacter spp. The highest arbekacin MIC among S. aureus isolates from PHP (43% methicillin-resistant S. aureus [MRSA]) was 4 µg/ml. Among P. aeruginosa isolates from PHP, only one had an arbekacin MIC of >16 µg/ml (MIC50 and MIC90, 1 and 4 µg/ml), and susceptibility rates for gentamicin, tobramycin, and amikacin were 88.0, 90.0, and 98.0%, respectively. Arbekacin (MIC50, 2 µg/ml) and tobramycin (MIC50, 4 µg/ml) were the most potent aminoglycosides tested against Acinetobacter baumannii. Against Enterobacteriaceae from PHP, arbekacin and gentamicin (MIC50 and MIC90, 0.25 to 1 and 1 to 8 µg/ml for both compounds) were generally more potent than tobramycin (MIC50 and MIC90, 0.25 to 2 and 1 to 32 µg/ml) and amikacin (MIC50 and MIC90, 1 to 2 and 2 to 32 µg/ml). Arbekacin also demonstrated potent in vitro activity against a worldwide collection of well-characterized MDR Gram-negative and MRSA strains.

[In vitro combined effects of double antibacterial drugs against multidrug-resistant Pseudomonas aeruginosa isolates: comparison among combinations of colistin, arbekacin, aztreonam, rifampicin and piperacillin].

This in vitro study examined the combined effects of double antibacterial drugs against multidrug-resistant Pseudomonas aeruginosa (MDRP). The tested clinical isolates from Hiroshima University Hospital were 40 strains which met the criteria for MDRP, that is, the minimum inhibitory concentration (MIC) was > or = 16 microg/mL of meropenem, > or = 4 microg/mL of ciprofloxacin and > or = 32 microg/mL of amikacin. Using the original checkerboard plates for colistin (CL), arbekacin (ABK), aztreonam (AZT), rifampicin (RFP) and piperacillin (PIPC), MIC values were determined for single and double combinations. Based on the MIC values, fractional inhibitory concentration index values were calculated and the combined effects (synergy action or additive action) were evaluated. The three strongest drugs among the tested combinations were i) CL + RFP (synergy, 80.0%; additive, 17.5%), ii) RFP + ABK (synergy, 7.5%; additive, 70.0%) and iii) RFP + AZT (synergy, 5.0%; additive, 77.5%). In these cases, the arithmetic mean MIC value of each drug significantly decreased as follows: i) 1.38 microg/mL (alone) and 0.26 microg/mL (with RFP) for CL, 19.85 microg/mL (alone) and 1.85 microg/mL (with CL) for RFP; ii) 19.85 microg/mL (alone) and 7.53 microg/mL (with ABK) for RFP, 8.87 microg/mL (alone) and 2.79 microg/mL (with RFP) for ABK; iii) 19.85 microg/mL (alone) and 10.15 microg/mL (with AZT) for RFP, 28.3 microg/ mL (alone) and 6.65 microg/mL (with RFP) for AZT. Of 40 strains, metallo-beta-lactamase and aminoglycoside 6'-N-acetyltransferase were found in 20 and 37 strains, respectively; however, no significant influence of these factors was observed on the combined effects of i), ii) and iii). The results of this study provide an in vitro rationale for RFP plus CL, ABK or AZT as an effective combination therapy for MDRP infections, although the results should be verified and compared with other antibacterial drugs in further studies.

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.

Determination of MIC distribution of arbekacin, cefminox, fosfomycin, biapenem and other antibiotics against gram-negative clinical isolates in South India: a prospective study.

OBJECTIVES: To determine the in vitro activity of antibiotics, including arbekacin, cefminox, fosfomycin and biapenem which are all still unavailable in India, against Gram-negative clinical isolates. METHODS: We prospectively collected and tested all consecutive isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. from blood, urine and sputum samples between March and November 2012. The minimum inhibition concentration (MIC) of 16 antibiotics was determined by the broth micro-dilution method. RESULTS: Overall 925 isolates were included; 211 E. coli, 207 Klebsiella spp., 153 P. aeruginosa, and 354 Acinetobacter spp. The MIC50 and MIC90 were high for cefminox, biapenem and arbekacin for all pathogens but interpretative criteria were not available. The MIC50 was categorized as susceptible for a couple of antibiotics, including piperacillin/tazobactam, carbapenems and amikacin, for E. coli, Klebsiella spp. and P. aeruginosa. However, for Acinetobacter spp., the MIC50 was categorized as susceptible only for colistin. On the other hand, fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while 90% of P. aeruginosa isolates were inhibited only by colistin. Finally, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested. CONCLUSION: Fosfomycin and colistin might be promising antibiotics for the treatment of infections due to E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India; however, clinical trials should first corroborate the in vitro findings. The activity of tigecycline should be evaluated, as this is commonly used as last-resort option for the treatment of multidrug-resistant Acinetobacter infections.

Emergence of Staphylococcus aureus carrying multiple drug resistance genes on a plasmid encoding exfoliative toxin B.

We report the complete nucleotide sequence and analysis of pETBTY825, a Staphylococcus aureus TY825 plasmid encoding exfoliative toxin B (ETB). S. aureus TY825 is a clinical isolate obtained from an impetigo patient in 2002. The size of pETBTY825, 60.6 kbp, was unexpectedly larger than that of the archetype pETBTY4 (∼30 kbp). Genomic comparison of the plasmids shows that pETBTY825 has the archetype pETBTY4 as the backbone and has a single large extra DNA region of 22.4 kbp. The extra DNA region contains genes for resistance to aminoglycoside [aac(6')/aph(2″)], macrolide (msrA), and penicillin (blaZ). A plasmid deletion experiment indicated that these three resistance elements were functionally active. We retrospectively examined the resistance profile of the clinical ETB-producing S. aureus strains isolated in 1977 to 2007 using a MIC determination with gentamicin (GM), arbekacin (ABK), and erythromycin (EM) and by PCR analyses for aac(6')/aph(2″) and msrA using purified plasmid preparations. The ETB-producing S. aureus strains began to display high resistance to GM, which was parallel with the detection of aac(6')/aph(2″) and mecA, after 1990. Conversely, there was no significant change in the ABK MIC during the testing period, although it had a tendency to slightly increase. After 2001, isolates resistant to EM significantly increased; however, msrA was hardly detected in ETB-producing S. aureus strains, and only five isolates were positive for both aac(6')/aph(2″) and msrA. In this study, we report the emergence of a fusion plasmid carrying the toxin gene etb and drug resistance genes. Prevalence of the pETBTY825 carrier may further increase the clinical threat, since ETB-producing S. aureus is closely related to more severe impetigo or staphylococcal scalded-skin syndrome (SSSS), which requires a general antimicrobial treatment.

The possibility of using fibrin-based collagen as an antibiotic delivery system.

PURPOSE: Collagen and fibrin are known to have potential use as a local drug-delivery system. This experimental study was designed to evaluate whether a fibrinogen-based collagen (FBC) fleece, coated with thrombin and aprotinin, can be used as an antibiotic delivery system. METHODS: In an in vitro study, gentamicin, fosfomycin, ampicillin, ciprofloxacin and dibekacin were absorbed by the FBC, Kirby-Bauer disks (KBDs), and expanded polytetrafluoroethylene. After washing with saline or phosphate buffer saline (PBS) 3 times for 6, 12 and 24 h, each sample was analyzed for antibiotic retention. In an in vivo study, we implanted the FBC onto mouse livers and dripped gentamicin and ciprofloxacin onto the FBC. The FBCs were subsequently collected and analyzed for their antibiotic activities. RESULTS: After irrigation with saline, each antibiotic showed different activities. After PBS washing, the FBC impregnated with each antibiotic had higher activity than the KBDs, and inhibited the bacterial growth by 60-80 % compared to the control. Gentamicin dripped onto the FBC could inhibit bacterial growth after 48 h in vivo without affecting the hemostatic properties of the FBC. However, the FBC treated with ciprofloxacin exhibited antibacterial activity for only 3 h. CONCLUSIONS: Some bases, including FBC, can retain antibacterial activities dependent on the ingredients of the base and the type of antibiotic. Gentamicin, but not ciprofloxacin, was retained in the FBC in vivo. These results suggest that absorbent FBC might be useful not only as hemostatic material, but also as a local drug-delivery system.

novel 6'-n-aminoglycoside acetyltransferase AAC(6')-Iaj from a clinical isolate of Pseudomonas aeruginosa.

Pseudomonas aeruginosa NCGM1588 has a novel chromosomal class 1 integron, In151, which includes the aac(6')-Iaj gene. The encoded protein, AAC(6')-Iaj, was found to consist of 184 amino acids, with 70% identity to AAC(6')-Ia. Escherichia coli transformed with a plasmid containing the aac(6')-Iaj gene acquired resistance to all aminoglycosides tested except gentamicin. Of note, aac(6')-Iaj contributed to the resistance to arbekacin. Thin-layer chromatography revealed that AAC(6')-Iaj acetylated all aminoglycosides tested except gentamicin. These findings indicated that AAC(6')-Iaj is a functional acetyltransferase that modifies the amino groups at the 6' positions of aminoglycosides and contributes to aminoglycoside resistance of P. aeruginosa NCGM1588, including arbekacin.

Evaluation of arbekacin anti-MRSA agents for adult in Japan.

There are a limited number of reports that compare the clinical efficacy of anti-MRSA agents such as arbekacin (ABK), vancomycin (VCM), teicoplanin (TEIC) and linezolid (LZD). There is a tendency for these four agents to show variation in the inflammatory response parameters, in C-reactive protein (CRP) and in white blood cell count (WBC), depending on the administration period. There was no significant difference among the agents in analysis of variance (ANOVA) in the group of days 1-3 (p = 0.0536) but there was some significant difference in the group of days 4-7, as well as days 8-14 (p < 0.001, p < 0.01) in relative variation rate of CRP. Furthermore, we compared in more detail the groups of LZD, VCM and ABK, with a significant decrease of CRP, each of which showed more decrease in comparison with the group of TEIC in the period days 4-7 (p < 0.01). We took 1-hr serum level after days 3-4, with the ABK treatment as the peak concentration (C(peak)). Having made nonlinear logistic regression analysis of CRP and C(peak)/MIC, we concluded that the decrease rate estimable by early inflammatory effect could be decreased to some 40%, assuming that C(peak)/MIC shows the high value within 4 days after ABK treatment.

[Clinical investigation of arbekacin sulfate based on Cmax/MIC].

We examined the peck concentration (Cmax)/minimal inhibitory concentration (MIC) and the clinical efficacy in methicillin-resistant Staphylococcus aureus (MRSA) pneumonia and Gram-positive cocci bacteremia. We evaluated arbekacin (ABK) on 22 cases of pneumonia and 10 cases of bacteremia in Aichi Medical University Hospital between August 2008 and July 2011, retrospectively. In pneumonia cases, Cmax/MIC was 16.4 +/- 2.8 in the effective group, and was 17.6 +/- 4.5 in the not effective group, the significant differences were not accepted (p = 0.8). The dosage of ABK was 4.7 +/- 1.4 mg/kg/dose in the effective group and was 4.3 +/- 0.7 mg/kg/dose in the not effective group. In bacteremia cases, Cmax/MIC was 24.2 +/- 13.9 in the effective group and 12.9 +/- 3.9 in the not effective group about clinical efficacy, and the high tendency was accepted by the effective group (p < 0.05). The dosage of ABK was 3.4 +/- 1.1 mg/kg/dose in the effective group, and 3.0 +/- 0.6 mg/kg/dose in the not effective group. In this examination, the significant difference was not observed in clinical efficacy and Cmax/MIC in the pneumonia cases. Although it was reported that clinical efficacy of ABK was given Cmax/MIC at eight or more, in this examination, all cases was eight or more at Cmax/MIC, and the clinical effect was 40.9%. On Cmax/MIC of ABK, clinical effective group was higher than not effective group in bacteremia cases, it was suggested that the administration design should make that Cmax/MIC at least about 14 or more would be necessary.

In vitro combination effects of aztreonam and aminoglycoside against multidrug-resistant Pseudomonas aeruginosa in Japan.

The aim of this study was to evaluate the in vitro combination effects of aztreonam (AZT) and aminoglycosides against multidrug-resistant (MDR) Pseudomonas aeruginosa strains in Japan. We investigated 47 MDR P. aeruginosa strains collected from 8 facilities. We selected the aminoglycosides amikacin (AMK), gentamicin (GM), and arbekacin (ABK) to examine their effects when combined with AZT using the checkerboard method. Of the 47 MDR P. aeruginosa strains, 41 tested positive for metallo-ß-lactamase (MBL). In all combinations, aminoglycosides decreased the minimum inhibitory concentrations of AZT in a dose-dependent manner, and there was no apparent antagonism. The combination effects were scored on a scale of 0 to 4, and statistical analysis was performed using the Wilcoxon signed-rank test. In all 47 strains, AZT + ABK (mean score, 2.02) had the highest score, followed by AZT + AMK (1.68) and AZT + GM (1.38) (ABK versus GM, P < 0.0001). In 41 MBL-positive strains, AZT + ABK (mean score, 2.05) had the highest score, followed by AZT + AMK (1.56) and AZT + GM (1.37) (ABK versus AMK, P = 0.02, and ABK versus GM, P < 0.0001). AZT + ABK was the most promising combination regimen against MDR P. aeruginosa strains; the other promising combinations were AZT + AMK and AZT + GM.

[Influence of inoculum size on MICs for methicillin-susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus].

Using 49 clinical methicillin-susceptible Staphylococcus aureus isolates (MSSA) and 54 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, we examined the change of MIC using five different inocula (2.5-4 x 10(2) cfu/spot-2.5-4 x 10(6) cfu/spot). We found the big change of the MIC with the increase of the inoculum size in ampicillin against MSSA, and the change was small in cefazolin, meropenem, ciprofloxacin. For anti-MRSA antibiotics, we found the small change with the increase of the inoculums size in vancomycin and arbekacin, and the middle change in teicoplanin and linezolid against MSSA and MRSA. The data from this study suggest that in serious and high inocula infections caused by S. aureus, the presence of an inoculum effect should be considered in curing.