NMR characterization of streptogramin B and L-156,587, a non-synergistic pair of the streptogramin family antibiotic complexes produced inductively by a combined culture of Streptomyces albogriseolus and Tsukamurella pulmonis.

The complete 1 H and 13 C NMR characterization of streptogramin B (1), the major component of a clinically important synergistic antibiotic complex, was presented for the first time, along with those of L-156,587 (2), a dehydrated congener of streptogramin A (3). Compounds 1 and 2 were not synergistic and produced by Streptomyces albogriseolus in co-culture with Tsukamurella pulmonis, which poses a question on the adaptive significance of the induced production of this antibiotic pair.

Modular Chemical Synthesis of Streptogramin and Lankacidin Antibiotics.

Continued, rapid development of antimicrobial resistance has become worldwide health crisis and a burden on the global economy. Decisive and comprehensive action is required to slow down the spread of antibiotic resistance, including increased investment in antibiotic discovery, sustainable policies that provide returns on investment for newly launched antibiotics, and public education to reduce the overusage of antibiotics, especially in livestock and agriculture. Without significant changes in the current antibiotic pipeline, we are in danger of entering a post-antibiotic era.In this Account, we summarize our recent efforts to develop next-generation streptogramin and lankacidin antibiotics that overcome bacterial resistance by means of modular chemical synthesis. First, we describe our highly modular, scalable route to four natural group A streptogramins antibiotics in 6-8 steps from seven simple chemical building blocks. We next describe the application of this route to the synthesis of a novel library of streptogramin antibiotics informed by in vitro and in vivo biological evaluation and high-resolution cryo-electron microscopy. One lead compound showed excellent inhibitory activity in vitro and in vivo against a longstanding streptogramin-resistance mechanism, virginiamycin acetyltransferase. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.Second, we recount our modular approaches toward lankacidin antibiotics. Lankacidins are a group of polyketide natural products with activity against several strains of Gram-positive bacteria but have not been deployed as therapeutics due to their chemical instability. We describe a route to several diastereomers of 2,18-seco-lankacidinol B in a linear sequence of ≤8 steps from simple building blocks, resulting in a revision of the C4 stereochemistry. We next detail our modular synthesis of several diastereoisomers of iso-lankacidinol that resulted in the structural reassignment of this natural product. These structural revisions raise interesting questions about the biosynthetic origin of lankacidins, all of which possessed uniform stereochemistry prior to these findings. Finally, we summarize the ability of several iso- and seco-lankacidins to inhibit the growth of bacteria and to inhibit translation in vitro, providing important insights into structure-function relationships for the class.

Synthetic group A streptogramin antibiotics that overcome Vat resistance.

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Potential RNA-dependent RNA polymerase inhibitors as prospective therapeutics against SARS-CoV-2.

Introduction. The emergence of SARS-CoV-2 has taken humanity off guard. Following an outbreak of SARS-CoV in 2002, and MERS-CoV about 10 years later, SARS-CoV-2 is the third coronavirus in less than 20 years to cross the species barrier and start spreading by human-to-human transmission. It is the most infectious of the three, currently causing the COVID-19 pandemic. No treatment has been approved for COVID-19. We previously proposed targets that can serve as binding sites for antiviral drugs for multiple coronaviruses, and here we set out to find current drugs that can be repurposed as COVID-19 therapeutics.Aim. To identify drugs against COVID-19, we performed an in silico virtual screen with the US Food and Drug Administration (FDA)-approved drugs targeting the RNA-dependent RNA polymerase (RdRP), a critical enzyme for coronavirus replication.Methodology. Initially, no RdRP structure of SARS-CoV-2 was available. We performed basic sequence and structural analysis to determine if RdRP from SARS-CoV was a suitable replacement. We performed molecular dynamics simulations to generate multiple starting conformations that were used for the in silico virtual screen. During this work, a structure of RdRP from SARS-CoV-2 became available and was also included in the in silico virtual screen.Results. The virtual screen identified several drugs predicted to bind in the conserved RNA tunnel of RdRP, where many of the proposed targets were located. Among these candidates, quinupristin is particularly interesting because it is expected to bind across the RNA tunnel, blocking access from both sides and suggesting that it has the potential to arrest viral replication by preventing viral RNA synthesis. Quinupristin is an antibiotic that has been in clinical use for two decades and is known to cause relatively minor side effects.Conclusion. Quinupristin represents a potential anti-SARS-CoV-2 therapeutic. At present, we have no evidence that this drug is effective against SARS-CoV-2 but expect that the biomedical community will expeditiously follow up on our in silico findings.

In vitro synergistic effect of retapamulin with erythromycin and quinupristin against Enterococcus faecalis.

To find a therapeutic alternative for the treatment of skin and soft tissue infections, we evaluated the effects of combinations of retapamulin with macrolide, lincosamide, and streptogramin (MLS) antibiotics against Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecium, and Enterococcus faecalis. Using both the disk diffusion test and checkerboard assay, we initially examined the effects of combinations of retapamulin with MLS antibiotics against standard strains of these species. Combinations of retapamulin with erythromycin, quinupristin/dalfopristin and quinupristin showed synergistic activity against E. faecalis only. Synergy of retapamulin with clindamycin and dalfopristin was not observed. Then, a checkerboard assay was performed to evaluate the effects of the combinations against 15 clinical strains of E. faecalis. Retapamulin and quinupristin, the most synergistic combination, showed activity against all erythromycin-susceptible, -intermediate, and -resistant strains tested. Among the eight strains with high-level erythromycin resistance, five strains were synergistically inhibited in the presence of only 1 µg of retapamulin per ml. Time-kill assay revealed that combinations of retapamulin with erythromycin and quinupristin were bacteriostatic. These results suggest that combinations of retapamulin with erythromycin and quinupristin have in vitro synergistic activity against E. faecalis, including strains with high-level erythromycin resistance.

Changes in Cognitive Processing Speed, Mood, and Fatigue in an Observational Study of Persons With Multiple Sclerosis Treated With Dalfampridine-ER.

OBJECTIVE: Multiple sclerosis (MS) is a degenerative neurological condition that results in impairments in multiple domains including cognition, fatigue, and mood. Dalfampridine-extended release (D-ER) has been approved to improve walking in persons with MS. It is plausible that D-ER could improve cognition, fatigue, and mood through the same mechanisms. We aim to examine effects of D-ER on cognition, depression, mood, and fatigue and to describe how these associations differ among those with and without D-ER related improvements in walking speed. METHODS: Patients with MS at the Mandell Center who were newly prescribed D-ER as part of their standard MS care were invited to participate in this observational pre-post study. Thirty-nine participants with MS were observed for 14 weeks; 31 remained on D-ER for 14 weeks or longer. Of these, 28 were then subdivided based on walk responder status. Cognition was assessed using the SDMT; depression was measured with the CESD. Self-reported cognition, mood, and fatigue were also measured using subscales of the Performance Scales (PS). RESULTS: Among those on drug through 14 weeks, there was significant improvement in the SDMT (P < 0.001) and the PS Fatigue score (P = 0.04). Among those who discontinued drug before 14 weeks, PS Cognition and PS Mood scores significantly improved (P = 0.02). Timed walk responders had significant improvements in SDMT (P < 0.001) and PS Fatigue (P = 0.046) from baseline to week 14. Among timed walk nonresponders, none of the measures significantly changed. CONCLUSIONS: Dalfampridine-extended release may improve cognition and fatigue in persons with MS, especially among timed walk responders.

Total synthesis of (-)-virginiamycin M2: application of crotylsilanes accessed by enantioselective Rh(II) or Cu(I) promoted carbenoid Si-H insertion.

A stereoselective synthesis of the antibiotic (-)-virginiamycin M(2) is detailed. A convergent strategy was utilized that proceeded in 10 steps (longest linear sequence) from enantioenriched silane (S)-15. This reagent, which was prepared via a Rh(II)- or Cu(I)-catalyzed carbenoid Si-H insertion, was used to introduce the desired olefin geometry and stereocenters of the C1-C5 propionate subunit. A modified Negishi cross-coupling or an efficient alkoxide-directed titanium-mediated alkyne-alkyne reductive coupling strategy was utilized to assemble the trisubstituted (E,E)-diene. An underutilized late-stage SmI(2)-mediated macrocyclization was employed to construct the 23-membered macrocycle scaffold of the natural product.

Pharmacogenomic strategies against microbial resistance: from bright to bleak to innovative.

The last decade saw an alarming increase in antibiotic resistance in infections, with more than 13 million deaths per year from infections. Counter strategies include hygiene, antibiotic restriction and new antibiotics such as quinupristin, linezolid, tigecycline, daptomycin and dalbavancin. Presently, pharmacogenomics with basic research is revealing new antimicrobial peptides and is applying old drugs in new ways to break resistance. New approaches with host-directed drug targeting emerge to circumvent resistance. A future systems perspective from large-scale molecular techniques and bioinformatic modeling allows pharmacogenomics to reveal new intervention angles. This includes the fight against resistance and its transmission, improved vaccines, disarmament of microbes and antibiotic options from novel molecular targets (lipids, RNA and carbohydrates). Such a system perspective is also essential for improved diagnostics and individualized medicine. However, an increase in public awareness and closer cooperation of industry and basic research are essential to turn research into powerful new drugs that will enable us to treat new arising infections in the future.

New treatment options and protocols for peritoneal dialysis-related peritonitis.

Peritonitis remains a major complication in patients undergoing peritoneal dialysis. The most recent ISPD guidelines for the empiric initial treatment of peritonitis recommend the use of antibiotics that provide coverage against Gram-positive organisms (vancomycin or cefazolin) and Gram-negative organisms (a third-generation cephalosporin or an aminoglycoside). However, there are some situations in which this regimen may not be desirable. Concerns of resistant organisms, changing microbiology, drug toxicity, or difficulties administering therapy may lead a provider to modify the initial regimen. Drug resistant Staphylococcus aureus strains and Enterococcus strains may require administration of newer agents such as linezolid, quinipristin/dalfopristin, or daptomycin. Many centers have reported that, over time, the microbiology at those institutions has been changing. Some centers have reported a significant decrease in gram positive organisms and increase in extended spectrum beta-lactamase (ESBL) organisms. It is important for each center to examine its microbiology to document such trends. Although the currently recommended therapies have low toxicities, it is possible that concerns for untoward side effects in an individual patient may dictate changing the regimen. Finally, there is evidence from many prospective studies that monotherapy with different agents (oral quinolones or cefepime) is efficacious; if ease of therapy is a consideration, these may also be appropriate agents.

[In vitro activities of quinupristin/dalfopristin in combination with vancomycin and gatifloxacin against Staphylococcus aureus and Enterococcus faecium clinical isolates]. / Staphylococcus aureus ve Enterococcus faecium klinik izolatlari üzerine kinupristin/dalfopristin ile vankomisin ve gatifloksasin kombinasyonlarinin in vitro aktiviteleri.

In this study, it was aimed to investigate the effects of quinupristin/dalfopristin in combination with vancomycin and gatifloxacin against Staphylococcus aureus and Enterococcus faecium isolates. A total 17 gram-positive bacterial isolates, composed of 4 methicillin-susceptible S. aureus (MSSA), 5 methicillin-resistant S. aureus (MRSA), 3 vancomycin-susceptible E. faecium (VSEF) and 5 vancomycin-resistant E. faecium (VREF) isolates, recovered from several clinical specimens in Ege University Faculty of Medicine, Turkey, were enrolled in this study. Antibiotic susceptibilities and interactions between antibiotics were determined by E-test (AB Biodisk, Sweden) method and fractional inhibitory concentration (FIC) indices were calculated for each combination. Synergistic activity was detected in only one MSSA isolate with the combination of quinupristin/dalfopristin and vancomycin (sigma FIC= 0.5). While the combination of quinupristin/dalfopristin and gatifloxacin yielded synergistic interaction in two MRSA and one MSSA isolate (sigma FIC= 0.37, 0.36 and 0.28, respectively) and additive interaction in one MSSA isolate (sigma FIC= 0.75), synergic activity was detected in one of the VREF isolate (sigma FIC= 0.29) and additive activity in two isolates (sigma FIC= 0.75 and 0.91, respectively). In this study, it was observed that the combination of quinupristin/dalfopristin and gatifloxacin was superior to the combination of quinupristin/dalfopristin and vancomycin especially in MRSA and VREF isolates. These in vitro results should be supported by in vivo studies which will guide the use of antibiotic combinations especially in the treatment of multi-resistant gram-positive bacterial infections.

A comparative in-vitro evaluation of resistance selection after exposure to teicoplanin, vancomycin, linezolid and quinupristin-dalfopristin in Staphylococcus aureus and Enterococcus spp.

The ability of breakpoint and serum concentrations of teicoplanin, vancomycin, linezolid and quinupristin-dalfopristin to select resistance was compared for isolates of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Enterococcus faecalis and Enterococcus faecium. Mutation frequencies were always <10(-10), except for two isolates grown in the presence of teicoplanin at the trough serum concentration. After multistep selection, linezolid selected for resistance in staphylococci and enterococci, and serial exposure to certain concentrations of linezolid was more likely to select for stable resistance in MRSA, MSSA and enterococci than was exposure to glycopeptides and quinupristin-dalfopristin.

De novo formal synthesis of (-)-virginiamycin M2 via the asymmetric hydration of dienoates.

A de novo approach to the formal total synthesis of the macrolide natural product (-)-virginiamycin M2 has been achieved via a convergent approach. The absolute and relative stereochemistry of the nonpeptide portion of (-)-virginiamycin M2 was introduced by two Sharpless asymmetric dihydroxylation reactions.

Transposon characterization of vancomycin-resistant Enterococcus faecium (VREF) and dissemination of resistance associated with transferable plasmids.

OBJECTIVES: VanA glycopeptide resistance has persisted on broiler farms in the UK despite the absence of the antimicrobial selective pressure, avoparcin. This study aimed to investigate the contribution of horizontal gene transfer of Tn1546 versus clonal spread in the dissemination of the resistance. METHODS AND RESULTS: One hundred and one vancomycin-resistant Enterococcus faecium isolated from 19 unrelated farms have been investigated. Tn1546 characterization by long PCR and ClaI-digestions of amplicons showed a very low diversity of Tn types (n=4) in comparison to the high genotypic diversity demonstrated by PFGE (n=62). Conjugation experiments were carried out to assess the transfer of vancomycin resistance. Co-transfer of vanA together with erm(B) positioned on the same conjugative plasmid containing a replicon similar to pRE25 was demonstrated and also the presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms. CONCLUSIONS: Horizontal transfer of vancomycin resistance may play a more important role in the persistence of antimicrobial resistance than clonal spread. The presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms, illustrates the ability of these enterococci to acquire and disseminate mobile genetic elements within integrated livestock systems.

Structural basis for streptogramin B resistance in Staphylococcus aureus by virginiamycin B lyase.

The streptogramin combination therapy of quinupristin-dalfopristin (Synercid) is used to treat infections caused by bacterial pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. However, the effectiveness of this therapy is being compromised because of an increased incidence of streptogramin resistance. One of the clinically observed mechanisms of resistance is enzymatic inactivation of the type B streptogramins, such as quinupristin, by a streptogramin B lyase, i.e., virginiamycin B lyase (Vgb). The enzyme catalyzes the linearization of the cyclic antibiotic via a cleavage that requires a divalent metal ion. Here, we present crystal structures of Vgb from S. aureus in its apoenzyme form and in complex with quinupristin and Mg2+ at 1.65- and 2.8-A resolution, respectively. The fold of the enzyme is that of a seven-bladed beta-propeller, although the sequence reveals no similarity to other known members of this structural family. Quinupristin binds to a large depression on the surface of the enzyme, where it predominantly forms van der Waals interactions. Validated by site-directed mutagenesis studies, a reaction mechanism is proposed in which the initial abstraction of a proton is facilitated by a Mg2+ -linked conjugated system. Analysis of the Vgb-quinupristin structure and comparison with the complex between quinupristin and its natural target, the 50S ribosomal subunit, reveals features that can be exploited for developing streptogramins that are impervious to Vgb-mediated resistance.

[Therapeutic perspectives of linezolid in the management of infections due to multiresistant Gram-positive pathogens]. / Le prospettive terapeutiche di linezolid nelle infezioni da patogeni Gram-positivi multiresistenti.

Multi-antibiotic resistant Gram-positive cocci represent emerging pathogens especially in the setting of the immunocompromised, hospitalized patients, in particular when surgery, invasive procedures, or prosthetic implants are of concern, patients are admitted in intensive care units, or underlying chronic disorders and immunodeficiency are of concern, and broad-spectrum antibiotics and/or immunosuppressive drugs are widely administered. The spectrum of available antimicrobial compounds for an effective management of these relevant infections is significantly impaired in selection and clinical efficacy by the emerging and spread of methicillin-resistant and more recently glycopeptide-resistant Gram-positive microbial strains linezolid, together with the recently licensed quinupristin-dalfopristin, daptomycin and tigecycline, followed by a number of glycopeptides, fluoroquinolones, and other experimental compounds represent an effective response to these concerns, due to their innovative mechanisms of action, their maintained or enhanced activity against multiresistant pathogens, their effective pharmacokinetic/pharmacodynamic properties, their frequent possibility of synergistic activity with other compounds effective against Gram-positive pathogens, and a diffuse potential for a safe and easy administration, also when compromised patients are of concern. The main problems related to the epidemiological and clinical features of multiresistant Gram-positive infection, the potential clinical indications of all recently available compounds compared with the standard of treatment of resistant Gram-positive infections, and updated data on efficacy and tolerability of linezolid have to be clarified.

In vitro activity of antimicrobial agents against oxacillin resistant staphylococci with special reference to Staphylococcus haemolyticus.

One hundred and sixty seven isolates of staphylococci isolated from the inpatients of a tertiary care referral hospital in South India were speciated and activity of oxacillin, glycopeptides, linezolid and quinupristin/dalfopristin against these isolates was tested by broth microdilution method. Of the 114 coagulase negative staphylococci (CoNS), 49.1 % were S. haemolyticus, isolated predominantly from urine (64.6%), while the rest belonged to 11 other species. More than half the isolates of S. aureus (52.8%) and 68.4% of the CoNS were oxacillin resistant. All the strains were uniformly susceptible to vancomycin, linezolid and quinupristin/dalfopristin; but 25.6% isolates of S. haemolyticus showed reduced susceptibility to teicoplanin (MIC: 8-16 mg/L). Our study demonstrates the high prevalence of oxacillin resistance among hospital isolates of S. aureus and CoNS in India. Vancomycin, along with the newer agents like linezolid and quinupristin/dalfopristin remains the drug of choice for treating multi drug resistant staphylococcal infections.

Prevalence of macrolide resistance genes among staphylococci in Cyprus.

The aims of the present study were to evaluate the frequency of macrolide-resistant staphylococci in Cyprus and to examine the phenotypic and genotypic characteristics of these isolates. Antimicrobial susceptibility testing was performed by broth microdilution method and the macrolide resistance determinants were detected by PCR. The relatedness among the isolates was examined by pulsed-field gel electrophoresis. Ninety-six (67.61%) of the 142 Staphylococcus aureus and 19 (59.4%) of the 32 coagulase-negative staphylococci were resistant to erythromycin. Among the 115 erythromycin-resistant staphylococci, 70 expressed the MLSB-inducible phenotype, 38 the MLSB-constitutive, and 7 the MS. The predominant genes associated with macrolide resistance were the ermA for S. aureus and the ermC for coagulase-negative staphylococci, detected in 90.62% and 47.37% of the isolates respectively. Dissemination of one clone carrying the ermA gene accounted for macrolide resistance in the majority of S. aureus isolates.