Search

BackgroundEnterococcus faecalis commonly produce aminoglycoside-modifying enzymes (AMEs) and are implicated in polymicrobial infections.ObjectivesTo determine if AME-producing E. faecalis is capable of protecting Enterobacteriaceae and Pseudomonas aeruginosa from gentamicin exposure.MethodsTwo Klebsiella pneumoniae isolates, two Escherichia coli isolates, and two Pseudomonas aeruginosa isolates were investigated in monoculture time–kill experiments, and each Gram-negative organism was also evaluated during co-culture with either AME-producing or AME-deficient E. faecalis. A pharmacokinetic/pharmacodynamics analysis that utilized Log Ratio Areas and a Hill-type mathematical model was used to determine if the maximal killing or potency of gentamicin against the Gram-negative organisms was altered by the presence of the E. faecalis.ResultsThe maximal killing and potency of gentamicin was the same during monoculture and co-culture experiments for both K. pneumoniae isolates and one E. coli isolate (P > 0.05). In contrast, the maximal killing of gentamicin was attenuated against one E. coli isolate and both P. aeruginosa isolates during co-culture with E. faecalis (P ConclusionsThe AME-producing E. faecalis did not provide a consistent protective effect from aminoglycosides for the Gram-negative pathogens.

Objectives Carbapenem-resistant Klebsiella pneumoniae (CR-KP) infections represent severe threats to public health worldwide. The aim of this study was to assess potential synergistic interaction between tigecycline and aminoglycosides via in vitro and in vivo studies. Methods Antibiotic resistance profiles and molecular characteristics of 168 CR-KP clinical isolates were investigated by susceptibility testing, PCR and MLST. Chequerboard tests and time–kill assays were performed for 20 CR-KP isolates to evaluate in vitro synergistic effects of tigecycline combined with aminoglycosides. A tissue-cage infection model of rats was established to evaluate in vivo synergistic effects. Different doses of tigecycline and aminoglycosides alone or in combination were administered for 7 days via tail vein injection. Antibiotic efficacy was evaluated in tissue-cage fluid and emergence of resistance was screened. Results The chequerboard tests showed that this combination displayed synergistic or partial synergistic activity against CR-KP. The time–kill assays further demonstrated that strong synergistic effects of such a combination existed against isolates that were susceptible to both drugs but for resistant isolates no synergy was observed if clinical pharmacokinetics were taken into consideration. The in vivo study showed that the therapeutic effectiveness of combination therapies was better than that of monotherapy for susceptible isolates, suggesting in vivo synergistic effects. Furthermore, combinations of tigecycline with an aminoglycoside showed significant activity in reducing the occurrence of tigecycline-resistant mutants. Conclusions Compared with single drugs, tigecycline combined with aminoglycosides could exert synergistic effects and reduce the emergence of tigecycline resistance. Such a combination might be an effective alternative when treating CR-KP infections in clinical practice.

Premature termination codon (PTC) readthrough is considered a potential treatment for genetic diseases caused by nonsense mutations. High concentrations of aminoglycosides induce low levels of PTC readthrough but also elicit severe toxicity. Identifying compounds that enhance PTC readthrough by aminoglycosides or reduce their toxicity is a continuing challenge. In humans, a binary complex of eukaryotic release factors 1 (eRF1) and 3 (eRF3a or eRF3b) mediates translation termination. They also participate in the SURF (SMG1-UPF1-eRF1-eRF3) complex assembly involved in nonsense-mediated mRNA decay (NMD). We show that PTC readthrough by aminoglycoside G418 is considerably enhanced by eRF3a and eRF3b siRNAs and cereblon E3 ligase modulators CC-885 and CC-90009, which induce proteasomal degradation of eRF3a and eRF3b. eRF3 degradation also reduces eRF1 levels and upregulates UPF1 and selectively stabilizes TP53 transcripts bearing a nonsense mutation over WT, indicating NMD suppression. CC-90009 is considerably less toxic than CC-885 and it enhances PTC readthrough in combination with aminoglycosides in mucopolysaccharidosis type I-Hurler, late infantile neuronal ceroid lipofuscinosis, Duchenne muscular dystrophy and junctional epidermolysis bullosa patient-derived cells with nonsense mutations in the IDUA, TPP1, DMD and COL17A1 genes, respectively. Combination of CC-90009 with aminoglycosides such as gentamicin or ELX-02 may have potential for PTC readthrough therapy.

Deleting the gene for small RNA GcvB in Escherichia coli was found to increase the sensitivity to several aminoglycoside antibiotics, such as neomycin, streptomycin, kanamycin, kasugamycin and spectinomycin, at low concentrations. GcvB, conserved in gram-negative enteric bacteria, is known to negatively control the expression of many genes for amino acid incorporation systems, especially the periplasmic ABC-transporter proteins. Deletions of several amino acid transporter genes in ΔgcvB cells decreased the antibiotic sensitivity to the wild-type level, suggesting that those genes are involved in uptake of aminoglycosides into the cell. Since GcvB is constitutively synthesized in growing cells, repressing synthesis of amino acid transporters, it contributes to the intrinsic resistance to several aminoglycoside antibiotics.

Mycobacterium avium (Mav) causes chronic infections in immunocompromised patients that require long-term antibiotic treatment. We have previously shown that Mav takes residence in host Mϕs and establishes a compartment (MavC) in which it is hidden from host defenses. Failure to establish the MavC traps Mav in Lamp1+ phagolysosomes where growth is prevented, and inflammatory signaling activated through TLRs 7/8. To elucidate how antibiotic treatment affects mycobacterial trafficking and host defenses, we infected human primary Mϕs with Mav for 4 days prior to treatment with a macrolide, aminoglycoside, and ethambutol. We show that Mav is killed and the MavC fuses with Lamp1+ lysosomes following antibiotic treatment. However, this does not result in nuclear translocation of NF-κB or production of inflammatory cytokines, suggesting different Lamp1+ lysosomal compartments can form that differ in their innate signaling capabilities. Thus, we show that upon antibiotic treatment of a chronic infection, Mav is quietly disposed of by Mϕs.

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.

ObjectivesTo characterize the genetic environment of the carbapenem resistance determinant in Proteus vulgaris of swine origin.MethodsThe carbapenem-resistant P. vulgaris strain BC22 was isolated from a faecal swab from a diseased pig with diarrhoea in Sichuan Province of China in 2018. The presence of carbapenemase genes was screened by PCR. WGS and bioinformatics analysis were performed to analyse the genetic environment of the carbapenem resistance determinant.ResultsP. vulgaris strain BC22 was found to harbour the carbapenemase gene blaNDM-1. WGS data revealed that blaNDM-1 was located in a truncated ISAba125 composite transposon. The carbapenem resistance gene blaNDM-1 and 20 other resistance genes, including the multiresistance gene cfr and the bifunctional aminoglycoside/quinolone resistance gene aac(6′)-lb-cr, were located in a novel SXT/R391 integrative and conjugative element (ICE). This new SXT/R391 ICE of 148.7 kb was chromosomally located, and could be transferred to Escherichia coli.ConclusionsHere, we report a carbapenemase gene, blaNDM-1, integrated into an SXT/R391 ICE. Our study highlights that this SXT/R391 ICE may facilitate the dissemination of clinically important resistance genes such as blaNDM-1, cfr and aac(6′)-lb-cr.

A list of our research achievements on multiple aminoglycoside antibiotic (AG) resistance in AG-producing actinomycetes is outlined. In 1979, the author discovered a novel AG (istamycin)-producing Streptomyces tenjimariensis SS-939 by screening actinomycetes with kanamycin (KM)-resistance and plasmid profiles. This discovery directed our biochemical and genetic approaches to multiple AG resistance (AGR) of AG producers. In this article, the following discoveries will be outlined: (1) AGR profiles correlating with the productivity of AGs in AG-producers, (2) Wide distribution of multiple AG resistance in AG-nonproducing actinomycetes, (3) Involvement of ribosomal resistance and AG-acetylating enzymes as underlying AGR factors, (4) Activation by single nucleotide substitution of a silent gene coding for aminoglycoside 3-N-acetyltransferase, AAC(3), in S. griseus, (5) Discovery of a novel antibiotic indolizomycin through protoplast fusion treatment between S. tenjimariensis and S. griseus strains with different AGR phenotypes, and (6) Double stage-acting activity of arbekacin (ABK; an anti-MRSA semisynthetic AG) discovered by acetylation of ABK with cloned AACs; that is both ABK and its acetylated derivatives showed remarkable antibiotic activities.

Host-directed therapies (HDTs) could enhance the activity of traditional antibiotics. AR-12 is a promising HDT against intracellular pathogens including Salmonella enterica serovar Typhimurium, and has been shown to act through modulation of autophagy and the Akt kinase pathway. Since AR-12 does not inhibit the growth of planktonic bacteria but only works in conjunction with the infected host-cell, we hypothesized that AR-12 could enhance the activity of antibiotics in less-susceptible strains in the intracellular host environment. We found that repetitive passaging of S. typhimurium in macrophages in the absence of antibiotics led to a 4-fold reduction in their intracellular susceptibility to streptomycin (STR), but had no effect on the bacteria's sensitivity to AR-12. Moreover, when the host-passaged strains were treated with a combined therapy of AR-12 and STR, there was a significant reduction of intracellular bacterial burden compared to STR monotherapy. Additionally, co-treatment of macrophages infected with multi-drug resistant S. typhimurium with AR-12 and STR or ampicillin showed enhanced clearance of the intracellular bacteria. The drug combination did not elicit this effect on planktonic bacteria. Overall, AR-12 enhanced the clearance of less susceptible S. typhimurium in an intracellular environment.

BackgroundCeftolozane/tazobactam is a novel cephalosporin/beta-lactamase inhibitor combination that often retains activity against resistant Pseudomonas aeruginosa. The comparative safety and efficacy vs polymyxins or aminoglycosides in this setting remains unknown.MethodsA retrospective, multicenter, observational cohort study was performed. Patients who received ceftolozane/tazobactam were compared with those treated with either polymyxin or aminoglycoside-based regimens for infections due to drug-resistant P. aeruginosa. Multivariate logistic regression was performed controlling for factors associated with treatment to assess the independent impact of ceftolozane/tazobactam on clinical cure, acute kidney injury (AKI), and in-hospital mortality.ResultsA total of 200 patients were included (100 in each treatment arm). The cohort represented an ill population with 69% in the intensive care unit, 63% mechanically ventilated, and 42% in severe sepsis or septic shock at infection onset. The most common infection type was ventilator-associated pneumonia (52%); 7% of patients were bacteremic. Combination therapy was more commonly used in polymyxin/aminoglycoside patients than those who received ceftolozane/tazobactam (72% vs 15%, P < .001). After adjusting for differences between groups, receipt of ceftolozane/tazobactam was independently associated with clinical cure (adjusted odds ratio [aOR], 2.63; 95% confidence interval [CI], 1.31–5.30) and protective against AKI (aOR, 0.08; 95% CI, 0.03–0.22). There was no difference in in-hospital mortality. The number needed to treat for a clinical cure with ceftolozane/tazobactam was 5, and the number needed to harm with AKI with a polymyxin/aminoglycoside was 4.ConclusionsThese data support the preferential use of ceftolozane/tazobactam over polymyxins or aminoglycosides for drug-resistant P. aeruginosa infections.

Background Trans-translation is a ribosome rescue system that plays an important role in bacterial tolerance to environmental stresses. It is absent in animals, making it a potential treatment target. However, its role in antibiotic tolerance in Pseudomonas aeruginosa remains unknown. Methods The role and activity of trans-translation during antibiotic treatment were examined with a trans-translation–deficient strain and a genetically modified trans-translation component gene, respectively. In vitro assays and murine infection models were used to examine the effects of suppression of trans-translation. Results We found that the trans-translation system plays an essential role in P. aeruginosa tolerance to azithromycin and multiple aminoglycoside antibiotics. We further demonstrated that gentamicin could suppress the azithromycin-induced activation of trans-translation. Compared with each antibiotic individually, gentamicin and azithromycin combined increased the killing efficacy against planktonic and biofilm-associated P. aeruginosa cells, including a reference strain PA14 and its isogenic carbapenem-resistance oprD mutant, the mucoid strain FRD1, and multiple clinical isolates. Furthermore, the gentamicin-azithromycin resulted in improved bacterial clearance in murine acute pneumonia, biofilm implant, and cutaneous abscess infection models. Conclusions Combination treatment with gentamicin and azithromycin is a promising strategy in combating P. aeruginosa infections.

BackgroundFosfomycin, effective in Cystic Fibrosis (CF), competes with aminoglycosides at renal binding sites and may therefore afford a renoprotective effect when used in combination therapy. We explored this by using markers of acute renal tubular damage [N-acetyl-β-d-glucose-aminidase (NAG), alanine amino-peptidase (AAP) and β2-microglobulin].MethodsUsing a prospective randomized crossover trial design, at an acute pulmonary exacerbation, 18 adult CF patients received either 14 days of intravenous (IV) tobramycin or IV tobramycin and IV fosfomycin, both in combination with a second IV antibiotic (colomycin).ResultsUrinary NAG (P = 0.003) and AAP (P = 0.03) following treatment with concomitant fosfomycin were lower than those after treatment with tobramycin and colomycin alone. Fosfomycin attenuated the total 24-h urinary protein leak (P = 0.0001). The 14-day improvements in all surrogate markers of exacerbation resolution (FEV1% predicted, FVC, white cell count and C-reactive protein) were similar for both treatment regimens.ConclusionThe addition of fosfomycin reduces acute renal injury caused by IV aminoglycoside therapy in CF pulmonary exacerbations.

Background The emergence of Neisseria gonorrhoeae resistant to all currently available antimicrobial therapies poses a dire public health threat. New antimicrobial agents with activity against N. gonorrhoeae are urgently needed. Apramycin is an aminocyclitol aminoglycoside with broad-spectrum in vitro activity against MDR Gram-negative pathogens and Staphylococcus aureus. However, its activity against N. gonorrhoeae has not been described. Objectives The activity spectrum of apramycin against a collection of MDR N. gonorrhoeae was assessed. Isolates tested included those susceptible and resistant to the structurally distinct aminocyclitol, spectinomycin. Results The modal MICs for apramycin and spectinomycin were 16 mg/L and 32 mg/L, respectively. The epidemiological cut-off (ECOFF) for apramycin was 64 mg/L. No strains among 77 tested had an MIC above this ECOFF, suggesting very low levels of acquired apramycin resistance. In time-kill analysis, apramycin demonstrated rapid bactericidal activity comparable to that of spectinomycin. Conclusions Apramycin has broad-spectrum, rapidly bactericidal activity against N. gonorrhoeae. Future pharmacokinetic and pharmacodynamic studies will be needed to determine whether apramycin and/or apramycin derivatives hold promise as new therapeutics for N. gonorrhoeae infection.

ObjectivesTo assess the ability of oxyclozanide to enhance tobramycin killing of Pseudomonas aeruginosa biofilms and elucidate its mechanism of action.MethodsTwenty-four hour biofilms formed by the P. aeruginosa strain PAO1 and cystic fibrosis (CF) isolates were tested for susceptibility to oxyclozanide and tobramycin killing using BacTiter-Glo™ and cfu. Biofilm dispersal was measured using crystal violet staining. Membrane potential and permeabilization were quantified using DiOC2(3) and TO-PRO-3, respectively.ResultsHere we show that the ionophore anthelmintic oxyclozanide, combined with tobramycin, significantly increased killing of P. aeruginosa biofilms over each treatment alone. This combination also significantly accelerated the killing of cells within biofilms and stationary phase cultures and it was effective against 4/6 CF clinical isolates tested, including a tobramycin-resistant strain. Oxyclozanide enhanced the ability of additional aminoglycosides and tetracycline to kill P. aeruginosa biofilms. Finally, oxyclozanide permeabilized cells within the biofilm, reduced the membrane potential and increased tobramycin accumulation within cells of mature P. aeruginosa biofilms.ConclusionsOxyclozanide enhances aminoglycoside and tetracycline activity against P. aeruginosa biofilms by reducing membrane potential, permeabilizing cells and enhancing tobramycin accumulation within biofilms. We propose that oxyclozanide counteracts the adaptive resistance response of P. aeruginosa to aminoglycosides, increasing both their maximum activity and rate of killing. As oxyclozanide is widely used in veterinary medicine for the treatment of parasitic worm infections, this combination could offer a new approach for the treatment of biofilm-based P. aeruginosa infections, repurposing oxyclozanide as an anti-biofilm agent.

Background\ud\udESBL-producing Escherichia coli have expanded globally since the turn of the century and present a major public health issue. Their in vitro susceptibility to penicillin/inhibitor combinations is variable, and clinical use of these combinations against ESBL producers remains controversial. We hypothesized that this variability related to co-production of OXA-1 penicillinase.\ud\ud\udMethods\ud\udDuring a national study we collected 293 ESBL-producing E. coli from bacteraemias, determined MICs by BSAC agar dilution, and undertook genomic sequencing with Illumina methodology.\ud\ud\udResults\ud\udThe collection was dominated by ST131 (n = 188 isolates, 64.2%) and blaCTX-M-15 (present in 229 isolates, 78.2%); over half the isolates (159/293, 54.3%) were ST131 with blaCTX-M-15. blaOXA-1 was found in 149 ESBL producers (50.9%) and blaTEM-1/191 in 137 (46.8%). Irrespective of whether all isolates were considered, or ST131 alone, there were strong associations (P

Background Antibiotics administered to farm animals have led to increasing prevalence of resistance genes in different microbiomes and environments. While antibiotic treatments help cure infectious diseases in farm animals, the possibility of spreading antibiotic resistance genes into the environment and human microbiomes raises significant concerns. Through long-term evolution, antibiotic resistance genes have mutated, thereby complicating the resistance problems. Results In this study, we performed deep sequencing of the gut microbiomes of 36 swine and 41 cattle in Korean farms, and metagenomic analysis to understand the diversity and prevalence of antibiotic resistance genes. We found that aminoglycoside, β-lactam, lincosamide, streptogramin, and tetracycline were the prevalent resistance determinants in both swine and cattle. Tetracycline resistance was abundant and prevalent in cattle and swine. Specifically, tetQ, tetW, tetO, tet32, and tet44 were the 5 most abundant and prevalent tetracycline resistance genes. Their prevalence was almost 100% in swine and cattle. While tetQ was similarly abundant in both swine and cattle, tetW was more abundant in swine than in cattle. Aminoglycoside was the second highest abundant resistance determinant in swine, but not in cattle. In particular, ANT(6) and APH(3′′) were the dominant resistance gene families in swine. β-lactam was also an abundant resistance determinant in both swine and cattle. Cfx was the major contributing gene family conferring resistance against β-lactams. Conclusions Antibiotic resistome was more pervasive in swine than in cattle. Specifically, prevalent antibiotic resistance genes (prevalence >50%) were found more in swine than in cattle. Genomic investigation of specific resistance genes from the gut microbiomes of swine and cattle in this study should provide opportunities to better understand the exchange of antibiotic resistance genes in farm animals.

Purpose The results of a study to determine the physical compatibility of plazomicin sulfate solution during simulated Y-site administration with 92 i.v. drugs are reported. Methods Plazomicin injection solution (500 mg/10 mL) was diluted in 0.9% sodium chloride or 5% dextrose for injection to a final volume of 50 mL (final plazomicin concentration, 24 mg/mL), consistent with a 15-mg/kg dose administered to an 80-kg patient (i.e., 1,200 mg). All other i.v. drugs were reconstituted according to manufacturers’ recommendations and diluted with 0.9% sodium chloride or 5% dextrose for injection to the upper range of concentrations used clinically. Y-site conditions were simulated by mixing 5 mL of plazomicin solution with 5 mL of tested drug solutions in a 1:1 ratio. Solutions were assessed for visual (via color and Tyndall beam testing), turbidity (using a laboratory-grade turbidimeter), and pH changes over a 60-minute observation period. Incompatibility was defined a priori as precipitation, color change, a positive Tyndall test, or a turbidity change of ≥0.5 nephelometric turbidity units at any time during the 60-minute observation period. Results Plazomicin was physically compatible with 79 of the 92 drugs tested. Determinations of physical incompatibility with plazomicin were made for 13 drugs: albumin, amiodarone, amphotericin B deoxycholate, anidulafungin, calcium chloride, daptomycin, esomeprazole, heparin, levofloxacin, methylprednisolone, micafungin, phenytoin, and propofol, Conclusion Plazomicin at a concentration of 24 mg/mL was physically compatible with 85% of the drugs tested, including 31 of 36 antimicrobial agents.

Objectives Rifampicin potentiates the activity of aminoglycosides (AGs) versus Pseudomonas aeruginosa by targeting the AmgRS two-component system. In this study we examine the impact of rifampicin on the AG susceptibility of cystic fibrosis (CF) lung isolates of P. aeruginosa and the contribution of AmgRS to AG resistance in these isolates. Methods amgR deletion derivatives of clinical isolates were constructed using standard gene replacement technology. Susceptibility to AGs ± rifampicin (at ½ MIC) was assessed using a serial 2-fold dilution assay. Results Rifampicin showed a variable ability to potentiate AG activity versus the CF isolates, enhancing AG susceptibility between 2- and 128-fold. Most strains showed potentiation for at least two AGs, with only a few strains showing no AG potentiation by rifampicin. Notably, loss of amgR increased AG susceptibility although rifampicin potentiation of AG activity was still observed in the ΔamgR derivatives. Conclusions AmgRS contributes to AG resistance in CF isolates of P. aeruginosa and rifampicin shows a variable ability to potentiate AG activity against these, highlighting the complexity of AG resistance in such isolates.

Background. There is little information about the efficacy of active alternative drugs to carbapenems except beta-lactam/beta-lactamase inhibitors for the treatment of bloodstream infections (BSIs) due to extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E). The objective of this study was to assess the outcomes of patients with BSI due to ESBL-E who received empiric therapy with such drugs (other active drugs [OADs]) or carbapenems. Methods. A multinational retrospective cohort study of patients with BSI due to ESBL-E who received empiric treatment with OADs or carbapenems was performed. Cox regression including a propensity score for receiving OADs was performed to analyze 30-day all-cause mortality as main outcome. Clinical failure and length of stay were also analyzed. Results. Overall, 335 patients were included; 249 received empiric carbapenems and 86 OADs. The most frequent OADs were aminoglycosides (43 patients) and fluoroquinolones (20 patients). Empiric therapy with OADs was not associated with mortality (hazard ratio [HR], 0.75; 95% confidence interval [CI],.38-1.48) in the Cox regression analysis. Propensity score-matched pairs, subgroups, and sensitivity analyses did not show different trends; specifically, the adjusted HR for aminoglycosides was 1.05 (95% CI,.51-2.16). OADs were neither associated with 14-day clinical failure (adjusted odds ratio, 0.62; 95% CI,.29-1.36) nor length of hospital stay. Conclusions. We were unable to show that empiric treatment with OAD was associated with a worse outcome compared with carbapenems. This information allows more options to be considered for empiric therapy, at least for some patients, depending on local susceptibility patterns of ESBL-E.

ispartof: JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY vol:75 issue:12 pages:3696-3697 ispartof: location:England status: published

Purpose The physical compatibility of commonly used agents that could be coadministered in the clinical setting with tedizolid phosphate during Y-site administration was evaluated. Methods Tedizolid phosphate vials were reconstituted to a final concentration of 0.8 mg/mL. All other drugs were prepared according to manufacturers’ recommendations and diluted with 0.9% sodium chloride injection (where applicable) to the highest standard concentrations used clinically. Y-site conditions were simulated in culture tubes by mixing 5 mL of tedizolid phosphate solution with 5 mL of the test drug solutions. The physical characteristics, turbidity, and pH of all admixtures were examined immediately after mixing and at 15, 60, and 120 minutes. Incompatibility was defined as gross precipitation, a positive Tyndall beam test, color changes, or increases in turbidity. Results With simulated Y-site administration, tedizolid phosphate was compatible with 69 of 86 drugs in 0.9% sodium chloride injection, including 24 of 31 antimicrobial agents. Of note, incompatibility was observed immediately after mixing except with ceftaroline and diphenhydramine, whose incompatibility with tedizolid phosphate was apparent after 15 and 60 minutes, respectively. Among the drug classes tested, tedizolid phosphate was compatible only with 1 aminoglycoside (amikacin) and incompatible with 1 echinocandin (caspofungin) and 1 cephalosporin (ceftaroline). In addition, tedizolid phosphate was incompatible with divalent cations (calcium chloride, calcium gluconate, and magnesium sulfate), probably due to precipitation with the phosphate component. A pH change of >1 unit occurred only with epinephrine (at 120 minutes). Conclusion Tedizolid phosphate 0.8 mg/mL in 0.9% sodium chloride injection was physically compatible with 69 of 86 study drugs during simulated Y-site administration.

Objective To evaluate augmented renal clearance (ARC) using aminoglycoside clearance (CLAMINO24h) derived from pharmacokinetic (PK) modelling. Methods A retrospective study at two paediatric hospitals of patients who received tobramycin or gentamicin from 1999 to 2016 was conducted. Compartmental PK models were constructed using the Pmetrics package, and Bayesian posteriors were used to estimate CLAMINO24h. ARC was defined as a CLAMINO24h of ≥130 mL/min/1.73 m2. Risk factors for ARC were identified using multivariate logistic regression. Results The final population model was fitted to 275 aminoglycoside serum concentrations. Overall clearance (L/h) was=CL0 × (TBW/70)0.75 × AGEH/(TMH + AGEH) + CL1 (0.5/SCr), where TBW is total body weight, H is the Hill coefficient, TM is a maturation term and SCr is serum creatinine. Median CLAMINO24h in those with versus without ARC was 157.36 and 93.42 mL/min/1.73 m2, respectively (P Conclusions ARC was observed in one of every five patients. Sepsis, increasing age and low SCr were associated with ARC. Increased clearance was associated with an attenuation of AUC24h in this population. Future studies are needed to define optimal dosing in paediatric patients with ARC.

Plazomicin (ACHN-490) is a novel parenteral aminoglycoside developed to target multidrug-resistant Enterobacteriaceae. It has recently been approved by the Food and Drug Administration for the management of complicated urinary tract infections and pyelonephritis caused by susceptible organisms. When compared with meropenem, plazomicin was not inferior. The adverse-event profile for plazomicin was comparable to meropenem except for an increased additional rise in serum creatinine in the plazomicin arm compared with the meropenem arm. This review focuses on the mode of action, antimicrobial activity, pharmacokinetics, clinical indications, and safety profile of this drug. Considerations for formulary addition and its place in therapy are also discussed.

Aims Pseudomonas aeruginosa can secrete large amounts of alginate during chronic infections and this has been associated with high resistance to antibiotics. The major aim of this study was to investigate whether degradation of extracellular alginate by alginate lyase would increase the sensitivity of Ps. aeruginosa to gentamicin, an aminoglycoside antibiotic. Methods and Results Degradation of alginate from Ps. aeruginosa was monitored using a spectrometric assay. Alginate lyase depolymerized alginate, but calcium and zinc cations at concentrations found in the cystic fibrosis lung reduced enzyme activity. Biofilms formed on agar were partially degraded by alginate lyase, but staining with crystal violet showed that the biomass of biofilms grown in liquid was not significantly affected by the enzyme. Viability testing showed that the sensitivity to gentamicin of biofilm bacteria and of bacteria released from biofilms was unaffected by alginate lyase. Conclusions Our results show that at least under the conditions used here alginate lyase does not affect gentamicin resistance of Ps. aeruginosa. Significance and Impact of the Study Our study indicates that alginate does not contribute to resistance to gentamicin and so does not provide support for the concept of treating patients with alginate lyase in order to increase the antibiotic sensitivity of Ps. aeruginosa.

OBJECTIVES The aac(6')-Ih gene encoding aminoglycoside 6'-N-acetyltransferase type I subtype h [AAC(6')-Ih] is plasmid-borne in Acinetobacter baumannii where it confers high-level amikacin resistance, but its origin remains unknown. We searched for the gene in the genomes of a collection of 133 Acinetobacter spp. and studied its species specificity, expression and dissemination. METHODS Gene copy number was determined by quantitative PCR, expression by quantitative RT-PCR, MIC by microdilution and transfer by plasmid mobilization. RESULTS The aac(6')-Ih gene was present in the chromosome of the two Acinetobacter gyllenbergii of the collection and was detected in all seven A. gyllenbergii clinical isolates. They had indistinguishable flanking regions indicating that the gene was intrinsic to this species. A. baumannii PIS Aba23 promoters were provided by insertion of ISAba23, which disrupted the Pnative promoter in A. gyllenbergii. Both types of promoters were similarly potent in Escherichia coli and A. baumannii. Aminoglycoside MICs for A. baumannii harbouring pIP1858 were higher than for A. gyllenbergii due to gene dosage. The non-self-transferable plasmid could be mobilized to other A. baumannii cells by the broad host range plasmid RP4. CONCLUSIONS We have found the origin of aac(6')-Ih in A. gyllenbergii, a species isolated, although rarely, in humans, and documented that dissemination of this gene is restricted to the Acinetobacter genus.

Should the Aminoglycoside β-Lactam Combination Be Abandoned in All Severely Ill Patients With Presumed Gram-Negative Infection?

A synthetic riboswitch N1, inserted into the 5′-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6′ hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6′-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

Background Plazomicin is a next-generation aminoglycoside that was developed to overcome common aminoglycoside-resistance mechanisms. Objectives We evaluated the activity of plazomicin and comparators against clinical isolates collected from 26 European and adjacent countries during 2014 and 2015 as part of the Antimicrobial Longitudinal Evaluation and Resistance Trends (ALERT) global surveillance programme. Methods All 4680 isolates collected from 45 hospitals were tested for susceptibility to antimicrobials using the reference broth microdilution method. Selected isolates were screened for genes encoding carbapenemases, aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases. Results Plazomicin (MIC50/90 0.5/2 mg/L) inhibited 95.8% of Enterobacteriaceae at ≤2 mg/L, including carbapenem-resistant Enterobacteriaceae (MIC50/90 0.25/128 mg/L). Plazomicin was more active compared with other aminoglycosides against isolates carrying blaKPC (MIC50/90 0.25/2 mg/L), isolates carrying blaOXA-48-like (MIC50/90 0.25/16 mg/L) and carbapenemase-negative isolates (MIC50/90 0.25/1 mg/L). Approximately 60% of the isolates harbouring blaVIM and blaNDM-1 carried 16S rRNA methyltransferases (mainly rmtB and armA). AME genes were detected among 728 isolates and 99.0% of these were inhibited by plazomicin at ≤2 mg/L. Plazomicin activity against Pseudomonas aeruginosa (MIC50/90 4/8 mg/L) was similar to amikacin activity (MIC50/90 2/16 mg/L). Plazomicin demonstrated activity against CoNS (MIC50/90 0.12/0.25 mg/L) and Staphylococcus aureus (MIC50/90 0.5/1 mg/L). Plazomicin activity was limited against Acinetobacter spp. (MIC50/90 8/>128 mg/L), Enterococcus spp. (MIC50/90 32/128 mg/L) and Streptococcus pneumoniae (MIC50/90 32/64 mg/L). Conclusions Plazomicin demonstrated activity against Enterobacteriaceae isolates tested in this study, including isolates carrying AMEs and a high percentage of the carbapenem-non-susceptible isolates. Plazomicin displayed activity against staphylococci.

Background Plazomicin is a next-generation aminoglycoside with in vitro activity against multidrug-resistant Gram-negative species, including carbapenem-resistant isolates. The Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE) is a federally funded, prospective multicenter consortium of 20 hospitals from nine US healthcare systems to track carbapenem-resistant Enterobacteriaceae. Methods Minimum inhibitory concentrations (MICs) of plazomicin were determined by broth microdilution according to current CLSI guidelines against a collection of 697 carbapenem-resistant Klebsiella pneumoniae with defined carbapenem resistance mechanisms, including KPC and OXA carbapenemases. Isolates were submitted by participating CRACKLE centers. Results Carbapenemases present in study isolates included KPC-2 (n = 323), KPC-3 (n = 364), KPC-4 (n = 2), OXA-48 like (n = 7), and NDM (n = 1). Plazomicin MICs ranged from ≤0.12 to >32 mg/L, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively (figure). MICs of 689 (98.8%) isolates were ≤4 mg/L, while MICs of the remaining eight isolates were >32 mg/L. Plazomicin MICs were related to specific carbapenemases present in isolates: of eight isolates with MICs >32 mg/L, seven contained OXA-48 like and one contained KPC-3, suggesting that these isolates possess an aminoglycoside-resistance mechanism on the same plasmid as their carbapenemase gene, such as a 16S ribosomal RNA methyltransferase, against which plazomicin is not active. Conclusion Plazomicin has good in vitro potency against a collection of carbapenemase-producing K. pneumoniae, with MIC90 value of 1 mg/L and MICs of ≤4 mg/L for 98.9% of isolates. Disclosures M. R. Jacobs, Achaogen: Investigator, Research grant. Shionogi: Investigator, Research grant. L. Connolly, Achaogen, Inc.: Consultant, Consulting fee. K. M. Krause, Achaogen: Employee, Salary. S. S. Richter, bioMerieux: Grant Investigator, Research grant. BD Diagnostics: Grant Investigator, Research grant. Roche: Grant Investigator, Research grant. Hologic: Grant Investigator, Research grant. Diasorin: Grant Investigator, Research grant. Accelerate: Grant Investigator, Research grant. Biofire: Grant Investigator, Research grant. D. Van Duin, achaogen: Scientific Advisor, Consulting fee. shionogi: Scientific Advisor, Consulting fee. Allergan: Scientific Advisor, Consulting fee. Astellas: Scientific Advisor, Consulting fee. Neumedicine: Scientific Advisor, Consulting fee. Roche: Scientific Advisor, Consulting fee. T2 Biosystems: Scientific Advisor, Consulting fee.

Purpose The advantages and disadvantages of once-daily versus conventional dosing of aminoglycoside antibiotics are reviewed. Summary Although administration of multiple daily doses remains the standard method of aminoglycoside dosing, once-daily dosing may provide enhanced clinical efficacy and reduced toxicity in selected patient populations; demonstrated pharmacokinetic and pharmacodynamic advantages include enhanced postantibiotic effect and an increased likelihood of a high ratio of serum peak concentration to minimum inhibitory concentration. Published evidence identified in a MEDLINE search covering the period 1985–2014 indicates that once-daily high-dose aminoglycoside therapy generally provides clinical effectiveness equivalent or superior to that of multiple-daily dosing. The risk of nephrotoxicity appears to be comparable with once- and multiple-daily aminoglycoside dosing. Several nomograms have been developed to facilitate once-daily dosing; the Hartford nomogram (using a dose of 7 mg/kg) is the most extensively tested and generally considered the most reliable. However, although those nomograms are convenient to use and may reduce expenses, a daily dosing regimen determined by individualized pharmacokinetic monitoring is likely to be more effective for achieving specific serum concentrations and may be a preferable approach for some patients. Patients who are pregnant or have liver failure, severe renal insufficiency, serious illness, or nutritional deficiency are not appropriate candidates for once-daily dosing. Conclusion Once-daily aminoglycoside dosing is an effective, well-established method to achieve therapeutic efficacy while limiting the risk of toxicity and simplifying the processes of dosing and monitoring.

UNLABELLED Growth of enteropathogenic Escherichia coli E2348/69 was inhibited by bicarbonate in a dose-dependent manner, showing approximately 5% growth reduction at 5 mmol l(-1) while kanamycin at 3·12 μg ml(-1) inhibited growth by 15%, yet when kanamycin and bicarbonate were combined at these concentrations, inhibition increased to 80%. Unexpectedly, at bicarbonate concentrations >20 mmol l(-1) enhancement of the antibiotic activity virtually disappeared, i.e. there was a paradoxical Eagle-like effect. How bicarbonate acts is unclear, but neutral or alkaline pH also enhanced the activity of kanamycin. However, several differences indicated a separate effect of bicarbonate. First, bicarbonate inhibited growth more than the corresponding increments in pH. Second, at low concentration, the antibiotic enhancing effect of bicarbonate was stronger than the effect of pH alone. Third, 5 mmol l(-1) bicarbonate significantly enhanced the activity of kanamycin while the corresponding pH had no effect. Fourth, the Eagle-like effect was exclusive of bicarbonate because changes in pH did not induce an analogous behaviour. Notwithstanding the mechanism, the enhancing effect of bicarbonate was indubitable. Consequently, it seems worthwhile to explore further its potential to improve the efficacy of aminoglycosides and maybe even other antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY Bicarbonate at a low concentration enhanced the in vitro antibiotic activity of kanamycin and gentamicin. Even though the action mechanism of bicarbonate is hitherto unknown, it seems worthwhile to explore further its capacity to improve the efficacy of aminoglycosides. Clearly, the well-known harmful side-effects of aminoglycosides are a concern. However, it has recently been shown in a fish model that bicarbonate may protect ciliary cells against the damage caused by aminoglycosides. So, it seems possible that bicarbonate could help reduce aminoglycoside dosage at the same time that it might help lessen the damage to auditory ciliary cells in humans.

G418, produced by fermentation of Micromonospora echinospora, is an aminoglycoside antibiotic commonly used in genetic selection and maintenance of eukaryotic cells. Besides G418, M. echinospora produces many G418 analogs. As a result, the G418 product always contains impurities such as gentamicin C1, C1a, C2, C2a, gentamicin A and gentamicin X2. These impurities are less potent but more toxic than G418, but the purification of G418 is difficult because it has similar properties to its impurities. G418 is an intermediate in the gentamicin biosynthesis pathway. From G418 the pathway proceeds via successive dehydrogenation and aminotransferation at the C-6′ position to generate the gentamicin C complex, but genes responsible for these steps are still obscure. Through disruption of gacJ, which is deduced to encode a C-6′ dehydrogenase, the biosynthetic impurities gentamicin C1, C1a, C2 and C2a were all removed, and G418 became the main product of the gacJ disruption strain. These results demonstrated that gacJ is in charge of conversion of the 6′-OH of G418 into 6′-NH2. Disruption of gacJ not only eliminates the impurities seen in the original strain but also improves G418 titers by 15-fold. G418 production was further improved by 26.6 % through traditional random mutagenesis. Through the use of combined traditional and recombinant genetic techniques, we produced a strain from which most impurities were removed and G418 production was improved by 19 fold.

Background In 2016 USCAST, the National Advisory Committee (NAC) for the United States (US) to EUCAST, undertook the re-evaluation of the in vitro susceptibility (AST) test interpretive criteria (IC) for gentamicin (GM), tobramycin (TO) and amikacin (AK) against Enterobacteriaceae (ENT), P. aeruginosa (PSA) and S. aureus (SA) based on an analysis of contemporary microbiology and PK/PD data. In 2019 USCAST posted the third version (www.uscast.org) of AG IC document and CLSI and EUCAST has published AG IC in CLSI M100-S29 and EUCAST v 9.0 documents. USCAST ICs for S were generally lower than those proposed by CLSI for all organism/drug combinations. PK/PD emphasized high, extended interval dosing (5 renal function groups) to reduce nephro-vestibular toxicity and a stasis exposure endpoint. Here, we evaluate the impact on S rates for US AST data that these IC changes created. Methods Clinical isolates from 2010 to 2018 US SENTRY Program (reference broth microdilution AST) were analyzed for S based on current and previous IC values. AG results for GM, TO and AK were evaluated against 66,280 ENT, 13,959 PSA and 51,950 SA. Benchmark S data for meropenem, cefepime, piperacillin–tazobactam and new AG, plazomicin (PZM) were included as well as ESBL and carbapenem-resistant ENT (CRE; 805 isolates). Results S rates for ENT as determined by USCAST IC were reduced by 4.2/1.2/3.1% for AK/GM/TO (CLSI) and by 3.3% for AK (EUCAST); no S rate difference for GM and TO as determined by USCAST/EUCAST. For PSA, S decreased by 46.8/6.2% for AK/TO (EUCAST) and 51.6/6.2% (CLSI). S for SA vs. GM declined by only 0.2% (CLSI). No AG IC could be calculated/offered for Acinetobacter or GM X PSA or AM/TO X SA. Best S overall coverage X ESBL (99.2%) or CRE (97.2%) isolates was by PZM. Conclusion USCAST IC updates for AG lead to reduced values for some organism/drug combinations among ENT and PSA compared with those proposed elsewhere. The USCAST-recommended ICs were based on achieving AUC/MIC ratio target associated with net bacterial stasis. Given the assumption of AG combination therapy, stasis was considered a reasonable endpoint when evaluating AG ICs to improve both safety and efficacy. Some organism X drug exposures could not be calculated and lower IC for pneumonia isolates (GM, TO) was recommended. Disclosures All authors: No reported disclosures.

Background Although aminoglycosides are recommended as part of empiric combination therapy in selected patients with healthcare-associated pneumonia, their efficacy and safety remains unclear. The objectives of this study were to evaluate the impact of empiric aminoglycoside treatment on microbiologic cure, recurrent pneumonia and death, and acute kidney injury (AKI) among hospitalized patients treated for pneumonia who were clinically cured. Methods This was a nested cohort study including 441 hospitalized subjects with confirmed bacterial pneumonia who achieved clinical cure. All subjects had positive respiratory cultures at the beginning of therapy and also had cultures obtained at the time of antibiotic completion. Subjects with the same pathogen present at both the beginning of and at the end of treatment were categorized as microbiologic failure and all others were categorized as microbiologic cure. Serum creatinine was measured at both the beginning and end of therapy, with an absolute increase in serum creatinine of 0.5 mg/L or greater defined as AKI. Composite outcomes of 30- and 90-day recurrent pneumonia or death following the clinical cure of the index pneumonia were captured. Patients who received empiric aminoglycoside therapy were compared with patients who did not receive aminoglycoside therapy. Results Of 441 included subjects, 14.5% (N = 64) received aminoglycoside therapy and 85.5% (N = 377) did not. The mean age was 54.7 years, with 70.5% male and 78.2% white. Characteristics of the two groups (including Charlson Comorbidity Indices and APACHE II scores) were similar. Rates of microbiologic cure, death/recurrent pneumonia at 30- and 90-days and AKI and were similar in both groups (table). In subgroup analyses restricted to different pathogen groups these associations remained unchanged. Conclusion Among hospitalized patients with pneumonia who were clinically cured, empiric aminoglycoside therapy was not associated with an increased likelihood of microbiologic cure, death or recurrent pneumonia or AKI. Disclosures All authors: No reported disclosures.

We report the case of a kidney-transplant patient, suffering an intra-abdominal abscess at the surgical site caused by a carbapenem-resistant ST258 Klebsiella pneumoniae clone, producing the KPC-3 carbapenemase. Under tigecycline treatment, the patient developed a sepsis caused by a carbapenem-susceptible ST258 K. pneumoniae strain. Complete DNA sequences of the plasmids carried by the resistant and susceptible strains from this patient were determined.The complete DNA sequences of plasmids were obtained by applying the 454 Genome Sequencer FLX-PLUS procedure on a library constructed of total plasmid DNA purified from the carbapenem-resistant and -susceptible strains.In the carbapenem-resistant strain, four plasmids encoding 24 resistance genes, including blaKPC-3, and two putative virulence clusters were detected. In the susceptible strain, large rearrangements occurred in the KPC-carrying plasmid, causing the deletion of the entire Tn4401::blaKPC-3 transposon, with the consequent reversion of the strain to carbapenem susceptibility. The patient was successfully treated with carbapenems and fully recovered.The description of the plasmid content in these two strains gives interesting insights into the plasticity of KPC-carrying plasmids in K. pneumoniae.

OBJECTIVES: To elucidate the role of the pcm-tolCsm operon in the multidrug resistance of Stenotrophomonas maltophilia. METHODS: The presence of the pcm-tolCsm operon was verified by RT-PCR. The phylogenetic relationship between the outer membrane proteins known to be involved in functional tripartite efflux in Escherichia coli, Pseudomonas aeruginosa and S. maltophilia was analysed. The contribution of TolCsm to resistance to a variety of compounds was investigated by susceptibility testing of the ΔtolCsm mutant. The role of pcm in the expression and function of tolCsm was assessed by quantitative real-time PCR and complementation assay. RESULTS: The pcm and tolCsm genes formed an operon. TolCsm of S. maltophilia, OpmH of P. aeruginosa and TolC of E. coli formed a distinguishing phylogenetic TolC-like clade. TolCsm deletion increased the susceptibility of S. maltophilia KJ2 to several antimicrobial agents (aminoglycoside, macrolide, β-lactam, chloramphenicol, nalidixic acid, doxycycline and trimethoprim/sulfamethoxazole) and chemical compounds (acriflavine, carbonyl cyanide 3-chlorophenylhydrazone, crystal violet, fusaric acid, menadione, Paraquat, plumbagin, SDS and tetrachlorosalicylanilide). The in-frame deletion of pcm caused a polar effect on the expression of tolCsm, which compromised the resistance to amikacin and gentamicin. Nevertheless, the presence of the PCM protein made an insignificant contribution to the function of TolCsm in the resistance to amikacin and gentamicin. CONCLUSIONS: The pcm-tolCsm operon makes a significant contribution to the multidrug resistance of S. maltophilia.

Received 10 August 2012; returned 4 October 2012; revised 6 February 2013; accepted 11 February 2013Objectives: This study examined the phenomenon of heteroresistance in Mycobacterium tuberculosis clinicalisolates obtained from retreated patients in Beijing, China between 2006 and 2011.Methods: The iPLEX Gold assay platform was used to determine the prevalence of heteroresistance to inject-able second-line drugs (amikacin, kanamycin and capreomycin) in resistant isolates.Results: Heteroresistance was identified in 10.9% of 220 phenotypic amikacin-resistant isolates.Conclusions: Heteroresistance was related mainly to the short duration and repeated use of amikacin andcapreomycin during retreatment. These findings further our understanding of the evolution of resistance to in-jectable drugs used for tuberculosis treatment and help guide the rational use of injectable drugs duringtherapy.Keywords: heteroresistance, MDR-TB, aminoglycoside antibiotics, molecular typing

Objectives Drug-induced kidney injury is a serious adverse event which needs to be monitored during aminoglycoside therapy. Urine cystatin C is considered an early and sensitive marker of nephrotoxicity. Cystatin C, a low-molecular-weight serum protein, and basic drugs have a common transport system expressed in the apical membrane of renal proximal tubular cells. The aim of this study was to investigate whether aminoglycoside antibiotics influenced cystatin C binding to the renal brush-border membrane. Methods The binding study was performed using a rapid filtration technique and affinity column displacement method. Key findings Concentration-dependent inhibition of chicken cystatin binding to brush-border membranes by gentamicin was observed. The gentamicin interaction with brush-border membranes was of relatively low affinity (Ki = 32 μm) in comparison with the chicken cystatin affinity to the binding sites (Kd = 3.6 μm). Amikacin and gentamicin were only able to displace chicken cystatin from the chromatographic affinity column in concentrations several times higher than normally found in the tubular fluid during standard aminoglycoside therapy. Conclusion Cystatin reabsorption in the proximal tubule cannot be significantly affected by aminoglycoside antibiotics because of their relatively low affinity to common binding sites on the brush-border membrane.

External guide sequences (EGSs) are short antisense oligoribonucleotides that elicit RNase P-mediated cleavage of a target mRNA, which results in inhibition of gene expression. EGS technology is used to inhibit expression of a wide variety of genes, a strategy that may lead to development of novel treatments of numerous diseases, including multidrug-resistant bacterial and viral infections. Successful development of EGS technology depends on finding nucleotide analogs that resist degradation by nucleases present in biological fluids and the environment but still elicit RNase P-mediated degradation when forming a duplex with a target mRNA. Previous results suggested that locked nucleic acids (LNA)/DNA chimeric oligomers have these properties. LNA are now considered the first generation of compounds collectively known as bridged nucleic acids (BNAs) – modified ribonucleotides that contain a bridge at the 2ʹ,4ʹ-position of the ribose. LNA and the second-generation BNA, known as BNANC, differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNANC and deoxynucleotide monomers in different configurations are nuclease resistant and could be excellent EGS compounds. However, not all configurations may be equally active as EGSs. RNase P cleavage assays comparing LNA/DNA and BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance aac(6ʹ)-Ib mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5ʹ- and 3ʹ-ends, respectively, were the most efficient EGSs while all BNANC/DNA gapmers showed very poor activity. When the most efficient LNA/DNA gapmer was covalently bound to a cell-penetrating peptide, the hybrid compound conserved the EGS activity as determined by RNase P cleavage assays and reduced the levels of resistance to amikacin when added to Acinetobacter baumannii cells in culture, an indication of cellular uptake and biological activity.

Background CRE are a world-wide public health challenge with extremely limited treatment options. Aminoglycosides have variable susceptibility against these organisms. At our institution, amikacin has been active against these isolates and useful as part of a combination regimen for CRE treatment. In this study, we compared the susceptibility results for 3 aminoglycosides between an automated susceptibility system (Phoenix) and broth microdilution (BMD). Methods Gentamicin, tobramycin, and amikacin susceptibility were determined in our academic medical center microbiology laboratory using an automated susceptibility system (Phoenix) and broth microdilution (BMD) according to CLSI guidelines against 120 recent CRE clinical isolates. Categorical agreement was defined between methods as classification of isolates in the same susceptibility category using CLSI breakpoints. Minor, major and very major error rates were calculated for each aminoglycoside. Results The primary CRE was K. pneumoniae (46%), followed by Enterobacter spp. (32%), and E. coli (6%). The categorical agreement ranged 58% (gentamicin) to 68% (tobramycin). Automated susceptibility system provided significantly higher susceptibility from 14% (gentamicin) to 30% (tobramycin and amikacin). Aminoglycoside Broth Microdilution Automated Susceptibility System %S MIC50 MIC90 MIC range %S MIC50 MIC90 MIC range Amikacin 63% 16 32 1 to >512 93% ≤8 16 ≤8 to >32 Tobramycin 12% 16 128 0.25 to >128 42% >8 >8 ≤2 to >8 Gentamicin 17% 16 128 0.25 to >128 31% >8 >8 ≤2 to >8 Aminoglycoside Categorical agreement Major error rate Very major error rate Minor error rate Amikacin 62.5% 0.8% 5.8% 30.8% Tobramycin 67.5% 0.8% 9.2% 22.5% Gentamicin 58.3% 4.2% 25.8% 11.7% Conclusion Automated susceptibility system over predicts the true susceptibility of CRE against all 3 aminoglycosides. This could be a major impact on the potential utility of the aminoglycosides especially amikacin for CRE infections. Disclosures All authors: No reported disclosures.

Aminoglycosides like gentamicin are among the most commonly used antibiotics in clinical practice and are essential for treating life-threatening tuberculosis and Gram-negative bacterial infections. However, aminoglycosides are also nephrotoxic and ototoxic. Although a number of mechanisms have been proposed, it is still unclear how aminoglycosides induce cell death in auditory sensory epithelia and subsequent deafness. Aminoglycosides bind to various intracellular molecules, such as RNA and phosphoinositides. We hypothesized that aminoglycosides, based on their tissue-specific susceptibility, also bind to intracellular proteins that play a role in drug-induced ototoxicity. By conjugating an aminoglycoside, gentamicin, to agarose beads and conducting a gentamicin-agarose pull-down assay, we have isolated gentamicin-binding proteins (GBPs) from immortalized cells of mouse organ of Corti, HEI-OC1. Mass spectrometry identified calreticulin (CRT) as a GBP. Immunofluorescence revealed that CRT expression is concentrated in strial marginal cells and hair cell stereocilia, primary locations of drug uptake and cytotoxicity in the cochlea. In HEI-OC1 cells treated with gentamicin, reduction of CRT expression using small interfering RNA (siRNA) reduced intracellular drug levels. CRT-deficient mouse embryonic fibroblast (MEF) cells as well as CRT siRNA-transfected wild-type MEFs also had reduced cell viability after gentamicin treatment. A pull-down assay using deletion mutants of CRT determined that the carboxyl C-domain of CRT binds to gentamicin. HeLa cells transfected with CRT C-domain deletion mutant construct were more susceptible to gentamicin-induced cytotoxicity compared with cells transfected with full-length CRT or other deletion mutants. Therefore, we conclude that CRT binding to gentamicin is protective against gentamicin-induced cytotoxicity.

Aims: The 2-deoxystreptamine-containing aminoglycoside antibiotics (AGAs) constitute the largest subgroup of the aminoglycosides. Neomycin (NEO) and lividomycin (LIV) are both representatives of the pseudo-tetrasaccharide group among the NEO-type AGAs. While NEO contains a 6′-NH2 group, the 6′-position remains unmodified in LIV. The aim of the study was to characterize the substrate specificities of the enzymes involved in the C-6′- and C-6‴-modification in order to explain the different amination patterns. Methods and Results: We overproduced and purified the enzymes NeoQ (bifunctional 6′- and 6‴-oxidoreductase) and NeoB (bifunctional 6′- and-6‴-aminotransferase), which had been analysed before (Huang et al. 2007), and compared the enzymatic properties with the corresponding enzymes LivQ (postulated 6‴-oxidoreductase, 72% identity to NeoQ) and LivB (postulated 6‴-aminotransferase, 71% identity to NeoB) from the LIV pathway. By applying a newly established photometric assay, we proved that LivQ oxidized only pseudotetrasaccharidic substrates at the 6‴-position. In contrast, NeoQ accepted also the pseudodisaccharidic paromamine as a substrate and oxidized the 6′- and 6‴-positions on two different precursors of NEO. The aminotransferases LivB and NeoB both transfer NH2 groups to the 6′-position in the precursor 6′-oxo-paromamine and to the 6‴-position of 6‴-oxo-neomycin C. Conclusions: The difference in the modification pattern of NEO and LIV at their 6′-positions is based only on the difference in the substrate specificities of the oxidoreductases LivQ and NeoQ, respectively. The aminotransferases LivB and NeoB share identical biochemical properties, and both are capable to transaminate the 6′ and also the 6‴-position of the tested AGAs. Significance and Impact of the Study: Our data provide information to understand the structural variations in aminoglycosides and may be helpful to interpret variations in other natural product bisoynthesis pathways.

Aminoglycosides offer several potential benefits in their treatment of peritoneal dialysis (PD)-associated peritonitis, including low cost, activity against Gram-negative organisms (including Pseudomonas aeruginosa), synergistic bactericidal activity against some Gram-positive organisms (such as Staphylococci) and relatively low propensity to promote antimicrobial resistance. However, there is limited conflicting evidence that aminoglycosides may accelerate loss of residual renal function (RRF) in PD patients. The aim of this study was to study the effect of aminoglycoside use on slope of decline in RRF.The study included 2715 Australian patients receiving PD between October 2003 and December 2007 in whom at least two measurements of renal creatinine clearance were available. Patients were divided according to tertiles of slope of RRF decline (rapid, intermediate and slow). The primary outcome was the slope of RRF over time in patients who received aminoglycosides for PD peritonitis versus those who did not.A total of 1412 patients (52%) experienced at least one episode of PD peritonitis. An aminoglycoside was used as the initial empiric antibiotic in 1075 patients. The slopes of RRF decline were similar in patients treated and not treated with at least one course of aminoglycoside (median [interquartile range] -0.26 [-1.17 to 0.04] mL/min/1.73 m(2)/month versus -0.22 [-1.11 to 0.01] mL/min/1.73 m(2)/month, P = 0.9). The slopes of RRF decline were also similar in patients receiving repeated courses of aminoglycoside.Empiric treatment with aminoglycoside for peritonitis was not associated with an adverse effect on RRF in PD patients.

Objectives: To examine the distribution and context of aminoglycoside resistance genes in multiply antibiotic-resistant Acinetobacter baumannii isolates from Australia that are members of the global clone 2 and carry the bla OXA-23 gene conferring resistance to carbapenems. Methods: Sixty-one multiply antibiotic-resistant A. baumannii strains isolated between 2000 and 2010 at six Australian hospitals that belonged to global clone 2 and carried the bla OXA-23 gene were studied. Various molecular techniques were used to determine their relatedness and to detect antibiotic resistance genes and insertion sequences. Structures surrounding the aminoglycoside resistance genes were sequenced. Results: The isolates all shared several antibiotic resistance genes, including the sul2 sulphonamide resistance gene, but varied in their pattern of resistance to aminoglycosides. The aminoglycoside resistance profiles of isolates were accounted for by four resistance genes-aadB, aacC1, aphA1b and aphA6-in various combinations. The aadB gene cassette was located at a secondary site on a 6 kb plasmid similar to pRAY. The aphA6 gene was in a transposon, TnaphA6, bounded by directly oriented copies of ISAba125. The aacC1 gene cassette in a class 1 integron and Tn6020 carrying aphA1b were always present together, but were not linked. Conclusions: Imipenem-resistant global clone 2 A. baumannii isolates containing bla OXA-23 have been present in Australian hospitals for at least 10 years. Variation in this global clone 2 type has occurred with the introduction of various aminoglycoside resistance genes carried on a small plasmid or within transposons.

BACKGROUND: Acute kidney injury is a relatively common complication of aminoglycoside therapy that affects 10-20% of patients receiving antibiotics of this class. Although the vast majority of patients do recover, the presence of certain risk factors may alter the clinical presentation and result in permanent renal damage. Thus, aminoglycoside-induced nephrotoxicity is a major concern and early diagnosis is critical. The aim of the present study was to characterize the early stages of aminoglycoside-induced nephrotoxicity using proton nuclear magnetic resonance ((1)H NMR) spectroscopy of urine. METHODS: We studied 19 previously healthy patients who were hospitalized in our clinic due to bacterial infections. Combined antibiotic treatment with amikacin (1 g once daily) and a beta-lactam antibiotic was instituted in all patients. Urine and blood samples were collected before and after 5 days of aminoglycoside treatment. RESULTS: (1)H NMR spectroscopic data showed increased amounts of alanine and lactic acid (+138 and +255% compared to baseline values, respectively) and decreased hippurate (-50%) after aminoglycoside treatment. In addition, fractional excretions of sodium, magnesium and calcium were significantly increased (+271, +295 and +60%, respectively). These findings indicate that aminoglycosides can affect nephron tubules through two unrelated mechanisms that produce a partial 'Fanconi-like syndrome' and a 'Bartter-like syndrome'. However, because none of the study participants developed renal failure, these alterations are probably reversible and should not be used as sensitive or specific indicators of impending renal insufficiency. CONCLUSIONS: Our study findings confirm that aminoglycosides can induce both proximal and distal renal tubular dysfunction. However, it remains unclear whether the early functional changes detected by (1)H NMR spectroscopy in patients treated with aminoglycosides are of prognostic value. Nephrol Dial Transplant