Structural basis for plazomicin antibiotic action and resistance.

The approval of plazomicin broadened the clinical library of aminoglycosides available for use against emerging bacterial pathogens. Contrarily to other aminoglycosides, resistance to plazomicin is limited; still, instances of resistance have been reported in clinical settings. Here, we present structural insights into the mechanism of plazomicin action and the mechanisms of clinical resistance. The structural data reveal that plazomicin exclusively binds to the 16S ribosomal A site, where it likely interferes with the fidelity of mRNA translation. The unique extensions to the core aminoglycoside scaffold incorporated into the structure of plazomicin do not interfere with ribosome binding, which is analogously seen in the binding of this antibiotic to the AAC(2')-Ia resistance enzyme. The data provides a structural rationale for resistance conferred by drug acetylation and ribosome methylation, i.e., the two mechanisms of resistance observed clinically. Finally, the crystal structures of plazomicin in complex with both its target and the clinically relevant resistance factor provide a roadmap for next-generation drug development that aims to ameliorate the impact of antibiotic resistance.

Structural and phylogenetic analyses of resistance to next-generation aminoglycosides conferred by AAC(2') enzymes.

Plazomicin is currently the only next-generation aminoglycoside approved for clinical use that has the potential of evading the effects of widespread enzymatic resistance factors. However, plazomicin is still susceptible to the action of the resistance enzyme AAC(2')-Ia from Providencia stuartii. As the clinical use of plazomicin begins to increase, the spread of resistance factors will undoubtedly accelerate, rendering this aminoglycoside increasingly obsolete. Understanding resistance to plazomicin is an important step to ensure this aminoglycoside remains a viable treatment option for the foreseeable future. Here, we present three crystal structures of AAC(2')-Ia from P. stuartii, two in complex with acetylated aminoglycosides tobramycin and netilmicin, and one in complex with a non-substrate aminoglycoside, amikacin. Together, with our previously reported AAC(2')-Ia-acetylated plazomicin complex, these structures outline AAC(2')-Ia's specificity for a wide range of aminoglycosides. Additionally, our survey of AAC(2')-I homologues highlights the conservation of residues predicted to be involved in aminoglycoside binding, and identifies the presence of plasmid-encoded enzymes in environmental strains that confer resistance to the latest next-generation aminoglycoside. These results forecast the likely spread of plazomicin resistance and highlight the urgency for advancements in next-generation aminoglycoside design.

Comparative in vitro activity of plazomicin and older aminoglyosides against Enterobacterales isolates; prevalence of aminoglycoside modifying enzymes and 16S rRNA methyltransferases.

Comparative in vitro activity of plazomicin and 4 older aminoglycosides was evaluated with broth microdilution in 714 blood isolates from 14 hospitals in Turkey. Isolates included Escherichia coli (n=320), Klebsiella spp. (n=294), Enterobacter spp. (n=69), Serratia marcescens (n=20), and Citrobacter spp. (n=11). Isolates resistant to older aminoglycosides (n=240) were screened for aminoglycoside modifying enzyme genes: aac(6')-Ib, aac(3)-Ia, aac(3)-IIa, ant(2″)-Ia. Isolates with high MICs for plazomicin (n=41) were screened for 16S rRNA methyltransferase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH, npmA) and 2 carbapenemase genes (blaOXA-48, blaNDM-1). Overall, resistance to plazomicin, amikacin, netilmicin, gentamicin, and tobramycin was 7.7%, 7.4%, 31.5%, 32.9%, and 34.7%, respectively. aac(6')-Ib and aac(3)-IIa were the most common AME genes. Co-occurrence of blaNDM-1 with armA and rmtC and blaOXA-48 with armA was striking. Enterobacter cloacae carrying rmtC+blaNDM-1, S. marcescens with armA+blaOXA-48, and rmtF+ blaOXA-48 in K. pneumoniae were reported for the first time.

In Vitro Packaging Mediated One-Step Targeted Cloning of Natural Product Pathway.

Direct cloning of natural product pathways for efficient refactoring and heterologous expression has become an important strategy for microbial natural product research and discovery, especially for those kept silent or poorly expressed in the original strains. Accordingly, the development of convenient and efficient cloning approaches is becoming increasingly necessary. Here we presented an in vitro packaging mediated cloning approach that combines CRISPR/Cas9 system with in vitro λ packaging system, for targeted cloning of natural product pathways. In such a scheme, pathways of Tü3010 (27.4 kb) and sisomicin (40.7 kb) were respectively cloned, and stuR was further depicted to positively regulate Tü3010 production. In vitro packaging mediated approach not only enables to activate cryptic pathways, but also facilitates refactoring or interrogating the pathways in conjunction with various gene editing systems. This approach features an expedited, convenient, and generic manner, and it is conceivable that it may be widely adopted for targeted cloning of the natural product pathways.

In-vitro activity of several antimicrobial agents against methicillin-resistant Staphylococcus aureus (MRSA) isolates expressing aminoglycoside-modifying enzymes: potency of plazomicin alone and in combination with other agents.

This study investigated the in-vitro activity of clinically relevant aminoglycosides and new antimicrobial agents-plazomicin, ceftobiprole and dalbavancin-against 55 methicillin-resistant Staphylococcus aureus (MRSA) isolates producing aminoglycoside-modifying enzymes (AMEs). The checkerboard method was used to assess synergism between plazomicin and four antibiotics (fosfomycin, ceftobiprole, cefoxitin and meropenem), and time-kill assays were performed for the most active combinations. Among the aminoglycosides tested, plazomicin was the most active agent against MRSA, with >90% of isolates being inhibited at a minimum inhibitory concentration (MIC) of ≤1 mg/L. MIC50 and MIC90 values for ceftobiprole and dalbavancin were 2 and 4 mg/L, and 0.125 and 0.125 mg/L, respectively. The most prevalent AME gene was aac(6')Ie-aph(2″)Ia (87.3%), followed by ant(4')Ia (52.7%) and aph(3')IIIa (52.7%). Plazomicin activity was not affected by the type or number of enzymes detected. In checkerboard and time-kill assays, indifference was the most common result achieved for the antibiotic combinations. Notably, no antagonism was observed with any combination tested. Overall, plazomicin in combination with meropenem had the highest synergistic effect, demonstrating synergy against seven isolates in the checkerboard assay and three isolates in time-kill curves. In conclusion, plazomicin showed potent activity against aminoglycoside-resistant MRSA isolates, regardless of the number and type of AMEs present. These findings indicate the potential utility of plazomicin in combination with meropenem for the treatment of MRSA infections.

Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase-VIa.

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3-acetyltransferase-VIa (AAC-VIa) are determined. Among the aminoglycoside N3-acetyltransferases, AAC-VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in kcat values. Larger differences in KM (∼40-fold) resulted in ∼30-fold variation in kcat /KM . Binding of aminoglycosides to AAC-VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside N3-acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H2 O and D2 O for the binary enzyme-sisomicin complex but remained the same in both solvents for the ternary enzyme-CoASH-sisomicin complex. Unlike, most other aminoglycoside-modifying enzymes, the values of ΔCp were within the expected range of protein-carbohydrate interactions. Solution behavior of AAC-VIa was also different from the more promiscuous aminoglycoside N3-acetyltransferases and showed a monomer-dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand-protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3-acetyltransferase. Proteins 2017; 85:1258-1265. © 2017 Wiley Periodicals, Inc.

Association between the Presence of Aminoglycoside-Modifying Enzymes and In Vitro Activity of Gentamicin, Tobramycin, Amikacin, and Plazomicin against Klebsiella pneumoniae Carbapenemase- and Extended-Spectrum-ß-Lactamase-Producing Enterobacter Species.

We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum ß-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL-, 6 KPC-/ESBL+, and 38 KPC-/ESBL- isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL- (25% and 38%, respectively), KPC-/ESBL+ (50% and 17%, respectively), and KPC-/ESBL- (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and <0.0001, respectively) or multiple AMEs (100% versus 13%, 33%, and 0%, respectively; P < 0.01 for all). KPC+/ESBL+ isolates also had a greater number of AMEs (mean of 4.6 versus 1.5, 0.9, and 0.05, respectively; P < 0.01 for all). GEN and TOB MICs were higher against isolates with >1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 µg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority.

Carbapenem-resistant Klebsiella pneumoniae strains exhibit diversity in aminoglycoside-modifying enzymes, which exert differing effects on plazomicin and other agents.

We measured in vitro activity of plazomicin, a next-generation aminoglycoside, and other aminoglycosides against 50 carbapenem-resistant Klebsiella pneumoniae strains from two centers and correlated the results with the presence of various aminoglycoside-modifying enzymes (AMEs). Ninety-four percent of strains were sequence type 258 (ST258) clones, which exhibited 5 ompK36 genotypes; 80% and 10% of strains produced Klebsiella pneumoniae carbapenemase 2 (KPC-2) and KPC-3, respectively. Ninety-eight percent of strains possessed AMEs, including AAC(6')-Ib (98%), APH(3')-Ia (56%), AAC(3)-IV (38%), and ANT(2")-Ia (2%). Gentamicin, tobramycin, and amikacin nonsusceptibility rates were 40, 98, and 16%, respectively. Plazomicin MICs ranged from 0.25 to 1 µg/ml. Tobramycin and plazomicin MICs correlated with gentamicin MICs (r = 0.75 and 0.57, respectively). Plazomicin exerted bactericidal activity against 17% (1× MIC) and 94% (4× MIC) of strains. All strains with AAC(6')-Ib were tobramycin-resistant; 16% were nonsusceptible to amikacin. AAC(6')-Ib combined with another AME was associated with higher gentamicin, tobramycin, and plazomicin MICs than AAC(6')-Ib alone (P = 0.01, 0.0008, and 0.046, respectively). The presence of AAC(3)-IV in a strain was also associated with higher gentamicin, tobramycin, and plazomicin MICs (P = 0.0006, P < 0.0001, and P = 0.01, respectively). The combination of AAC(6')-Ib and another AME, the presence of AAC(3)-IV, and the presence of APH(3')-Ia were each associated with gentamicin resistance (P = 0.0002, 0.003, and 0.01, respectively). In conclusion, carbapenem-resistant K. pneumoniae strains (including ST258 clones) exhibit highly diverse antimicrobial resistance genotypes and phenotypes. Plazomicin may offer a treatment option against strains resistant to other aminoglycosides. The development of molecular assays that predict antimicrobial responses among carbapenem-resistant K. pneumoniae strains should be a research priority.

Analysis of sisomicin binding sites in Micromonospora inyoensis cell wall.

Sisomicin binding sites are located in the cell wall. They are the carboxyl groups of peptidoglycans, which are major components of the cell wall. The carboxyl groups have negative charges which can bind the positively charged amino groups of sisomicin. When the negative charges of the carboxyl groups of the peptidoglycans are changed to neutral or positive charges, sisomicin does not bind to the cell wall. Magnesium ions bind to the cell wall in competition with sisomicin, and have a weak affinity for the cell wall binding sites compared with sisomicin. The binding molar ratio of sisomicin to magnesium ions was approximately 1 to 10.

[Determination of sisomicin in body fluid by high performance liquid chromatography-indirect photometric detection method].

A high performance liquid chromatography-indirect photometric detection (HPLC-IPD) method for determination of sisomicin sulfate in body fluid (serum, urine, saliva, cerebrospinal fluid and fluid oozed out from wound) was established. The conditions in this method were a mobile phase solution of methyl alcohol-acetonitrile-water (20:10:70) containing nicotinamide 0.5 mmol/L, sodium 1-heptanesulfonate 5 mmol/L and phosphoric acid 0.05 mol/L, a Spherisorb C18 column (4.6 mm x 250 mm) and an UV detector with sensitivity of 0.05 Aufs (0.1 Aufs for urine). The detection wavelength was fixed at 268 nm. The average recovery for sisomicin in serum was 96.92% +/- 4.63% and the coefficients of variation were 4.75% and 5.65% for within-day and day-to-day tests respectively. The detectable limit was 0.1 ng. The concentration of sisomicin in body fluid of 4 patients was determined.

Human pharmacology of 5-epi-sisomicin (Sch 22591) following intramuscular administration.

5-epi-sisomicin was given as a single intramuscular injection of 1 mg/kg to six healthy male adults. Serum peak concentrations averaged 3.07 mg/l, the mean elimination half life was 179 min and the mean 24 h urinary recovery was 85.3%. Local and systemic tolerance was good.

[Principles for analyzing the kinetic curves of the antimicrobial effect in dynamic systems simulating the pharmacokinetic profiles of antibiotics]. / Printsipy analiza krivykh kinetiki antimikrobnogo éffekta v dinamicheskikh sistemakh, modeliruiushchikh farmakokineticheskie profily antibiotikov.

Alternative variants of the available methods for estimating the antimicrobial effect kinetics in the in vitro dynamic systems were analyzed. For defining and analyzing the concentration-effect relations in the in vitro dynamic systems it was recommended that two integral parameters characterizing the antimicrobial effect duration (TE) and intensity (IE) irrespective of the recording means be used. TE is defined by the time from the moment of antibiotic administration into the dynamic system till the moment when the count of the microorganisms reaches again its initial level. IE is defined by the area between the curves of the microbial growth kinetics in the presence and absence of an antibiotic. The possible application of TE and IE is exemplified by relation between the sisomicin antimicrobial effect on P. aeruginosa 58, E. coli 93 and K. pneumoniae 5056 and the antibiotic concentration under conditions of sisomicin pharmacokinetic profiles reproduction after intramuscular administration within the levels of the therapeutic doses with an account of individual variability of the aminoglycoside concentration in the blood of humans.

[Interrelations in the changes in the pharmacokinetics and nephrotoxic effect of sisomycin as affected by cefazolin]. / Vzaimosviaz' izmenenii v farmakokinetike i nefrotoksicheskom éffekte sizomitsina pod vliianiem tsefazolina.

The action of cefazolin on the pharmacokinetics and nephrotoxic effect of sisomicin was studied on Wistar rats. Sisomicin in doses of 12.5 and 25 mg/kg alone or in combination with cefazolin in doses of 90 and 360 mg/kg was administered intramuscularly to the animals daily for 16 days. It was shown that in both the doses cefazolin had no noticeable action on the level of the functional and morphological changes in the kidneys. Consequently, there were no significant changes in the levels of sisomicin in serum and the site of the nephrotoxic effect (cortical layer of the kidneys) and in the half-life of the aminoglycoside in the kidney cortical layer under the action of cefazolin. At the same time there was observed a marked individual variability of the levels of urea nitrogen and sisomicin in serum of the rats treated with the aminoglycoside alone or in combination with cefazolin. Analysis of the dependence of the nephrotoxic effect on concentration of sisomicin in serum after its use alone or in combination with cefazolin revealed that the changes in the individual intensity of the effect in all the cases were mainly induced by the changes in the sisomicin blood levels. Therefore, control of the blood levels of the aminoglycoside should provide prevention of the development of its nephrotoxic effect not only in monotherapy but also in the use of aminoglycosides in combination with cefazolin.

[Clinical evaluation of sisomicin in the treatment of complicated chronic urinary tract infections administered by intravenous infusion].

The pharmacokinetics of sisomicin (SISO) were determined in 3 patients including a case with mild renal dysfunction after intravenous infusion over 1 hour of a single dose of 50 mg. The peak serum concentration was ranged from 3.3 to 3.9 micrograms/ml with about 2 hours of half-life in patients with normal renal function and 4.5 hours in a patient with renal impaired function. This result suggested that dosage regimen should be adjusted in patients with renal impaired function. Clinical response was evaluated in treatment of 16 cases with chronic complicated UTI. SISO was administered intravenously over 1 or 1.5 hours at a daily dose of 100 or 150 mg. An overall excellent or moderate effect was seen in 68.8% of treatment cases with the evaluation by the UTI committee's criteria. As for clinical laboratory abnormal values, a slight but reversible increase of BUN was observed in 1 case. In conclusion, intravenous administration of SISO appeared to be effective and useful method for treatment of complicated UTI.

[Sisomycin pharmacokinetics in the perilymph and blood serum--an approach to predicting its ototoxic effect]. / Izuchenie farmakokinetiki sizomitsina v perilimfe i syvorotke krovi--podkhod k prognozirovaniiu ototoksicheskogo éffekta.

To elucidate the possibility of predicting the level of aminoglycoside antibiotic penetration into the fluids of the internal ear by the antibiotic blood levels, the pharmacokinetics of sisomicin in the perilymph and blood serum was studied on guinea pigs. The antibiotic was administered to the animals subcutaneously in doses of 50, 100 and 200 mg/kg. On the basis of the comparison of the sisomicin concentrations in the perilymph normalized against the dose it was concluded that the pharmacokinetics of sisomicin in the perilymph and blood serum of the animals was linear. Comparison of the areas under the curves of the antibiotic concentration versus time in the perilymph (AUCp) and blood serum (AUCs) showed that the tissue availability of the antibiotic in this study characterized by its penetration into the perilymph and defined by the ratio of the AUCp to AUCs amounted to 55 per cent. In a two-compartment model it was not possible to predict the antibiotic levels in the perilymph by concentrations in the blood. However, by the antibiotic blood levels it was possible to characterize in a complex the pharmacokinetic behaviour of the antibiotic in the perilymph by predicting the areas under the respective curves of the antibiotic concentration versus time. The proportional relation between the values of the AUCp and AUCs suggested that the level of the antibiotic penetration into the internal ear and consequently the intensity of the potential ototoxic effect could be more reliably predicted not by separate values of the antibiotic concentration but by the areas under curves of aminoglycoside concentrations versus time.

The single-dose pharmacokinetics of 5-epi-sisomicin (Sch 22591) in human volunteers.

5-epi-sisomicin was given as a single 1 mg/kg intramuscular injection to six adult male volunteers. No adverse effects were observed and the pharmacology was very similar to that of sisomicin. The extended spectrum of 5-epi-sisomicin and its enhanced antipseudomonal activity prompt further clinical evaluation of this agent.

[Pharmacokinetics of sisomicin during hemosorption and hemodialysis]. / Osobennosti farmakokinetiki sizomitsina pri gemosorbtsii i gemodialize.

The effect of hemosorption and hemodialysis on the pharmacokinetics of sisomicin was studied in 17 patients with acute and chronic renal insufficiency. The value of the antibiotic extraction coefficient in hemosorption was almost 2 times higher than that in hemodialysis. In patients on hemosorption, extracorporeal elimination of the antibiotic was of the saturation nature. It was characterized by systematic diminishing of the extraction coefficient, while in patients on hemodialysis, it did not depend on the time (the value of the extraction coefficient was constant). In this connection it is recommended that the rate of diminishing of the extraction coefficient in hemosorption be estimated. Since sisomicin is rapidly absorbed by the column it is not advisable to administer sisomicin to patients before hemosorption.

[Decrease of sisomicin nephrotoxicity as affected by cephalothin: pharmacokinetic evaluation]. / Oslablenie nefrotoksichnosti sizomitsina pod vliianiem tsefalotina: popytka farmakokineticheskogo ob'iasneniia.

The effect of cephalothin on the nephrotoxicity and pharmacokinetics of sisomicin was studied on Wistar rats. Sisomicin was injected intramuscularly in doses of 12.5 and 25 mg/kg alone or in combination with cephalothin in a dose of 360 mg/kg once a day for 16 days. It was shown that the combined use of sisomicin and cephalothin resulted in less pronounced functional and morphological changes in the kidneys as compared to the use of sisomicin alone. The decrease in the nephrotoxic effect was accompanied by a decrease in the sisomicin concentration in the blood serum and the site of the nephrotoxic effect (the kidney cortical layer) and the period of the aminoglycoside half-life in the kidney cortical layer under the action of cephalothin. The analysis of the relation between the nephrotoxic effect and the concentration of sisomicin in the kidney cortical layer and blood serum demonstrates that the nephrotoxicity of the sisomicin combination with cephalothin is mainly due to a decrease in the aminoglycoside concentration in the zone of the nephrotoxic effect.

[A dynamic system for the in vitro study of the kinetics of the antimicrobial effect of antibiotics in pharmacokinetic changes in their concentration]. / Dinamicheskaia sistema dlia izucheniia kinetiki antimikrobnogo éffekta in vitro pri farmakokineticheski obuslovlennykh izmeneniiakh kontsentratsii antibiotika.

The present approaches to in vitro investigation of the kinetics of the antibiotic antimicrobial effect are based on its recording at one or another constant concentration of the drugs. The regularities revealed under such conditions do not usually reflect the peculiar effect of the drugs in vivo, since with the use of the routine schemes of dosing the drug levels in the body fluids always vary. For investigation of the kinetics of the antimicrobial effect of antibiotics at their varying levels, a universal dynamic system providing in vitro simulation of the pharmacokinetic profiles of the drugs in blood and tissues was developed. The system is based on the controlled dilution of the medium containing an antibiotic and microbial cells. the dynamic system allows simulation of any kinetic profile of the drugs with regard to the constants of the linear pharmacokinetic N-compartmental model. The trial of the dynamic system as applied to the antimicrobial effect of sisomicin on strain A 20 363 of E. coli revealed significant differences in the effect of the drug, when its bioexponential pharmacokinetic profile was simulated in a one-compartmental model with exponential absorption and a constant concentration of the antibiotic. The check determinations of the antibiotic concentration in the main flask of the system showed that the system provided high accuracy simulation of the required antibiotic levels.