Important Class Of Antibiotics Research Articles

First Y-type actinomycins from Streptomyces with divergent structure-activity relationships for antibacterial and cytotoxic properties

Streptomyces sp. strain Gö-GS12 was found to produce five novel actinomycins Y(1)-Y(5) (). Their amino acid pattern discloses them as members of a new family of this important class of antibiotics. Compounds differ from Z-type actinomycins in their beta-peptidolactone rings which here contain trans-4-hydroxyproline (Hyp) or 4-oxoproline (OPro) amino acids, and from the X-congeners by containing methylalanine (MeAla). Within the new Y-type actinomycins variations are not only in the rare chlorinated or hydroxylated threonine residue. Furthermore, the beta-ring can undergo rearrangement by a two-fold acyl shift (compounds and ) or show a unique additional ring closure with the chromophore (compound ), resulting in metabolites with yet unknown structural motifs, altered conformations and distinct bioactivities. The strongest bioactivity was found for the chlorine containing actinomycin Y(1) (), the most surprising for Y(5) () with cytotoxic and antibacterial effects losing their coherence, which has been observed for the first time here.

Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary

Glycopeptide antibiotics have long served as drugs of last resort for the treatment of antibiotic-resistant gram-positive bacterial infections. Resistance to the clinically relevant glycopeptides, vancomycin and teicoplanin, threatens to undermine the usefulness of this important class of antibiotics. DNA extracted from a geographically diverse collection of soil samples was screened by PCR for the presence of sequences related to OxyC, an oxidative coupling enzyme found in glycopeptide biosynthetic gene clusters. Every soil sample examined contained at least 1 unique OxyC gene sequence. In an attempt to access the biosynthetic gene clusters associated with these OxyC sequences, a 10,000,000-membered environmental DNA (eDNA) megalibrary was created from a single soil sample. Two unique glycopeptide gene clusters were recovered from this eDNA megalibrary. Using the teicoplanin aglycone and the 3 sulfotransferases found in one of these gene clusters, mono-, di-, and trisulfated glycopeptide congeners were produced. The high frequency with which OxyC genes were found in environmental samples indicates that soil eDNA libraries are likely to be a rewarding source of glycopeptide gene clusters. Enzymes found in these gene clusters should be useful for generating new glycopeptides analogs. Environmental DNA megalibraries, like the one constructed for this study, can provide access to many of the natural product biosynthetic gene clusters that are predicted to be present in soil microbiomes.

Structural basis for hygromycin B inhibition of protein biosynthesis.

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.

The Expanding Role of Lipid II as a Target for Lantibiotics

Lipid II is an essential cell-wall precursor required for the growth and replication of both Gram-positive and Gram-negative bacteria. Compounds that use lipid II to selectively target bacterial cells for destruction represent an important class of antibiotics. Clinically, vancomycin is the most important example of an antibiotic that operates in this manner. Despite being considered the 'antibiotic drug of last resort', significant bacterial resistance to vancomycin now manifests itself worldwide. In this paper we review recent progress made in understanding the lipid II-associated antibacterial characteristics of various naturally occurring compounds, with particular focus on the lantibiotic peptides.

Resistenza ai fluorochinoloni in Escherichia coli isolati da infezioni delle vie urinarie (IVU) in pazienti ospedalizzati in unità di terapia intensiva (UTI).

Background: Fluoroquinolones are an important class of antibiotics for the treatment of urinary tract infections that have axcellent activity against Escherichia coli, one of the most frequently encountered pathogens. Several European studies have reported an increase of resistance to quinolones among uropathogenic E. coli. We conducted this study to update our knowledge on this evolution. Materials and methods: We evaluated the resistance phenotype of 203 clinical strains of E. coli collected from urine specimens. The following antimicrobial agents were tested: ampicillin, amoxiciclin-clavulanate, piperacillintazobactam, cefamandole, cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, trimethoprimsulfamethoxazole, gentamicin, amikacin, ciprofloxacin, pipemidic acid, norfloxacin, nitrofurantoin. Disk diffusion tests were carried out as suggested by the CLSI (2006); strains were assigned to the susceptibility categories (susceptible, intermediate and resistant) interpreting results according to the established breakpoints. Results: Resistance to quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was about of 45% and resistance to ampicillin, trimethoprim-sulfamethoxazole was 57. 1% and 55. 2%, respectively. Resistance rates less than 5% was found for cefepime, amikacin and imipenem. Conclusions: This study confirms the evolution through resistance to quinolones of uropathogenic E. coli isolates. The selective pressure exerted by fluoroquinolones may influence this evolution. Therapeutic alternatives, surveillance, and restriction of fluoroquinolones use are needed to control this spread of resistance.

A Novel Lantibiotic Acting on Bacterial Cell Wall Synthesis Produced by the Uncommon Actinomycete Planomonospora sp.

Important classes of antibiotics acting on bacterial cell wall biosynthesis, such as beta-lactams and glycopeptides, are used extensively in therapy and are now faced with a challenge because of the progressive spread of resistant pathogens. A discovery program was devised to target novel peptidoglycan biosynthesis inhibitors capable of overcoming these resistance mechanisms. The microbial products were first screened according to their differential activity against Staphylococcus aureus and its L-form. Then, activities insensitive to the addition of a beta-lactamase cocktail or d-alanyl-d-alanine affinity resin were selected. Thirty-five lantibiotics were identified from a library of broth extracts produced by 40,000 uncommon actinomycetes. Five of them showed structural characteristics that did not match with any known microbial metabolite. In this study, we report on the production, structure determination, and biological activity of one of these novel lantibiotics, namely, planosporicin, which is produced by the uncommon actinomycete Planomonospora sp. Planosporicin is a 2194 Da polypeptide originating from 24 proteinogenic amino acids. It contains lanthionine and methyllanthionine amino acids generating five intramolecular thioether bridges. Planosporicin selectively blocks peptidoglycan biosynthesis and causes accumulation of UDP-linked peptidoglycan precursors in growing bacterial cells. On the basis of its mode of action and globular structure, planosporicin can be assigned to the mersacidin (20 amino acids, 1825 Da) and the actagardine (19 amino acids, 1890 Da) subgroup of type B lantibiotics. Considering its spectrum of activity against Gram-positive pathogens of medical importance, including multi-resistant clinical isolates, and its efficacy in vivo, planosporicin represents a potentially new antibiotic to treat emerging pathogens.

Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis

Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.

Aminoglycoside Resistance by ArmA-mediated Ribosomal 16 S Methylation in Human Bacterial Pathogens

Aminoglycosides are a medically important class of antibiotics used to treat serious infections. Methylation of the ribosomal target is an emerging mechanism that produces a high level of resistance to all clinically available aminoglycosides for systemic therapy except streptomycin. ArmA was the first methyltransferase using this mechanism to be discovered in a clinical isolate. We demonstrate that ArmA methylates the N7 position of nucleotide G1405 in 16S rRNA. Methylation at this position is presumed to mediate cellular resistance by blocking aminoglycoside binding by ribosomes. To test this hypothesis, we measured the binding of gentamicin by 30S subunits. Under our conditions, we did not observe binding by ribosomes methylated by ArmA. Furthermore, the ArmA methylation reaction is specific for the 30S ribosomal subunit; neither 16S rRNA alone nor the 70S ribosome is a substrate for this reaction under our experimental conditions, implicating ribosomal proteins in substrate recognition. The biochemical characteristics of ArmA place it in the Agr family of methyltransferases, whose members are predominantly anti-suicide genes from Actinomycetes aminoglycoside producers. The discrepancy between the 30% GC content of armA and the >60% GC content of Actinomycetes, however, calls into question the origin of armA. We demonstrate that surprisingly, the natural promoter of armA from gram-negative Klebsiella pneumoniae was active in gram-positive Bacillus subtilis, suggesting that armA originated from a low-GC, gram-positive aminoglycoside-producing organism.

Current Antibiotic Therapy for Isolated Urinary Tract Infections in Women

Sulfa antibiotics, such as a combination product of trimethoprim and sulfamethoxazole, have traditionally been the drugs of choice for urinary tract infections (UTIs) and remained the most common treatment as recently as a decade ago. However, increasing sulfa resistance among Escherichia coli may have led to changes in prescribing practices. We used the 2000-2002 National Ambulatory Medical Care Survey and National Hospital Ambulatory Medical Care Survey to obtain nationally representative data on antibiotics prescribed for women with isolated outpatient UTIs following visits to physicians' offices, hospital clinics, and emergency departments (n = 2638). Logistic regression was used to determine predictors of quinolone use. Quinolones were more commonly prescribed than sulfa antibiotics in each year evaluated. In the most recent year of data, quinolones were prescribed in 48% and sulfas in 33% of UTI visits (P<.04). Quinolones were significantly more likely to be prescribed to older patients and in visits occurring in the Northeast; however, no difference in quinolone prescribing was seen when evaluating insurance status, setting, race, ethnicity, health care provider type, and year. Approximately one third of the quinolones used were broader-spectrum agents. Quinolones have surpassed sulfas as the most common class of antibiotic prescribed for isolated outpatient UTI in women. Few significant predictors of quinolone use exist, suggesting that the increase is not confined to a certain subset of patients. This pervasive growth in quinolone use raises concerns about increases in resistance to this important class of antibiotics.

Molecular Evolution Perspectives on Intraspecific Lateral DNA Transfer of Topoisomerase and Gyrase Loci in Streptococcus pneumoniae , with Implications for Fluoroquinolone Resistance Development and Spread

Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.

A New Route to Designer Antibiotics

AbstractFor Abstract see ChemInform Abstract in Full Text.

A New Route to Designer Antibiotics

To overcome resistance, antibiotics must be modified regularly, but their complex structures make this a daunting task. In their Perspective, [Khosla and Tang][1] discuss how chemical synthesis and biosynthesis can complement each other in this quest for more powerful future antibiotics. They highlight the report by [ Charest et al ][2]., who have devised a powerful synthetic strategy for assembling novel tetracyclines, an important class of antibiotics that is widely used in humans and animals. Synthetic and biosynthetic approaches allow different parts of the tetracycline molecule to be modified with ease, and by combining the two approaches, these different strengths can be harnessed to find better antibiotics. [1]: http://www.sciencemag.org/cgi/content/full/308/5720/367 [2]: http://www.sciencemag.org/cgi/content/short/308/5720/395

Regulation and biosynthesis of carbapenem antibiotics in bacteria

Carbapenem antibiotics are members of the beta-lactam family of antibiotics, the most important class of antibiotics currently in clinical use. They are active against many important Gram-positive and Gram-negative pathogens. One important feature of carbapenem antibiotics is their resistance to several beta-lactamases. Thienamycin, isolated from Streptomyces cattleya, was the first carbapenem described. Other well-studied carbapenems were isolated from the Gram-negative bacteria Erwinia carotovora subsp. carotovora, Serratia sp. strain ATCC39006 and Photorhabdus luminescens strain TT01. Here, we review the genetics and biochemistry of carbapenem production in these bacteria. Research into carbapenems could uncover a new repertoire of bioactive molecules and biosynthetic enzymes, and exploiting these novel enzymes could lead to development of new classes of antibiotics with useful chemotherapeutic activities.

Binding site of macrolide antibiotics on the ribosome: new resistance mutation identifies a specific interaction of ketolides with rRNA.

Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of macrolide antibiotics.

MexXY-OprM Efflux Pump Is Required for Antagonism of Aminoglycosides by Divalent Cations in Pseudomonas aeruginosa

Antagonism of aminoglycosides by divalent cations is well documented for Pseudomonas aeruginosa and is regarded as one of the problems in aminoglycoside therapy. It is generally considered that divalent cations interfere with uptake of aminoglycosides at both the outer and inner membranes. It has been demonstrated recently that aminoglycosides can be removed from cells of P. aeruginosa by the three-component multidrug resistance efflux pump MexXY-OprM. We sought to investigate the interplay between efflux and uptake in resistance to aminoglycosides in P. aeruginosa. To do so, we studied the effects of the divalent cations Mg(2+) and Ca(2+) on susceptibility to aminoglycosides in a wild-type strain of P. aeruginosa and in mutants either overexpressing or lacking the MexXY-OprM efflux pump. MICs of gentamicin, streptomycin, amikacin, apramycin, netilmicin, and arbekacin were determined in Mueller-Hinton broth in the presence of cations added at concentrations that varied from 0.125 to 8 mM. We found, unexpectedly, that while both Mg(2+) and Ca(2+) antagonized aminoglycosides (up to a 64-fold decrease in susceptibility at 8 mM), antagonism was seen only in the strains of P. aeruginosa that contained the functional MexXY-OprM efflux pump. Our results indicate that inhibition of the MexXY-OprM efflux pump should abolish the antagonism of aminoglycosides by divalent cations, regardless of its precise mechanism. This may significantly increase the therapeutic index of aminoglycosides and improve the clinical utility of this important class of antibiotics.

Deoxysugars in glycopeptide antibiotics: enzymatic synthesis of TDP-L-epivancosamine in chloroeremomycin biosynthesis.

The 2,3,6-trideoxysugar l-epivancosamine is the terminal sugar added to the aglycone scaffold in chloroeremomycin, a member of the vancomycin family of glycopeptide antibiotics. Five proteins from the chloroeremomycin biosynthetic cluster, ORF14 and ORF23 to ORF26, have been expressed heterologously in Escherichia coli and purified to near homogeneity, and each has been characterized for an enzymatic activity. These five enzymes reconstitute the complete biosynthesis of TDP-l-epivancosamine from TDP-4-keto-6-deoxy-d-glucose. This process involves C-2 deoxygenation, C-3 amination and methylation, C-5 epimerization, and C-4 ketoreduction. Intermediates and the final product of this pathway have been identified by mass spectrometry and NMR. The pathway established here represents the complete in vitro reconstitution of an unusual sugar for an important class of antibiotics and sets the groundwork for future combinatorial biosynthesis for new bioactive compounds.

Recognition of a cognate RNA aptamer by neomycin B: quantitative evaluation of hydrogen bonding and electrostatic interactions.

Aminoglycosides are an important class of antibiotic that selectively target RNA structural motifs. Recently we have demonstrated copper derivatives of amino-glycosides to be efficient cleavage agents for cognate RNA motifs. To fully develop their potential as pharmaceutical agents it is necessary to understand both the structural mechanisms used by aminoglycosides to target RNA, and the relative contributions of hydrogen bonding and electrostatic interactions to recognition selectivity. Herein we report results from a calorimetric analysis of a stem-loop 23mer RNA aptamer complexed to the aminoglycoside neomycin B. Key thermodynamic parameters for complex formation have been determined by isothermal titration calorimetry, and from the metal-ion dependence of these binding parameters the relative contributions of electrostatics and hydrogen bonding toward binding affinity have been assessed. The principal mechanism for recognition and binding of neomycin B to the RNA major groove is mediated by hydrogen bonding.

Origins of the aminoglycoside modifying enzymes

The aminoglycosides represent an important class of antibiotics for the treatment of bacterial infections. Interaction of the aminoglycosides with the bacterial ribosome inhibits protein synthesis, which is their primary mode of action. However, in gram negative bacteria, the ability of aminoglycosides to perturb the cell envelope is also an important mode of action. A common mechanism for aminoglycoside resistance involves modifying enzymes which acetylate, phosphorylate or adenylylate the aminoglycoside. There exists a staggering number of aminoglycoside modifying enzymes in clinical isolates of bacteria. This diversity suggests multiple origins for the present day enzymes. In this review, the possible origins of these modifying enzymes will be presented. Previous proposals describing the origins of these enzymes will be reviewed and potential mechanisms for the development of new aminoglycoside modifying enzymes will be discussed.

Yeast glycosylation mutants are sensitive to aminoglycosides.

Aminoglycosides are a therapeutically important class of antibiotics that inhibit bacterial protein synthesis and a number of viral and eukaryotic functions by blocking RNA-protein interactions. Vanadate-resistant Saccharomyces cerevisiae mutants with defects in Golgi-specific glycosylation processes exhibit growth sensitivity to hygromycin B, an aminoglycoside [Ballou, L., Hitzeman, R. A., Lewis, M. S. & Ballou, C. E. (1991) Proc. Natl. Acad. Sci. USA 88, 3209-3212]. Here, evidence is presented that glycosylation is, in and of itself, a key factor mediating aminoglycoside sensitivity in yeast. Examination of mutants with a wide range of glycosylation abnormalities reveals that all are sensitive to aminoglycosides. This effect is specific to aminoglycosides and not merely a consequence of increased permeability of the yeast mutants to drugs. Furthermore, inhibition of glycosylation in wild-type cells leads to a marked increase in their sensitivity to aminoglycosides. These results establish that a defect in glycosylation is sufficient to render yeast cells susceptible to these clinically important drugs. Further, they suggest that a molecule which prevents the uptake or mediates removal of aminoglycosides requires glycosylation for its activity. Perhaps more importantly, this finding on drug sensitivity provides the most powerful screen to date to identify mutants and thereby to isolate genes involved in all aspects of N-linked glycosylation.

Update on glycopeptide resistance in enterococci

The glycopeptides vancomycin and teicoplanin constitute an important class of antibiotics for the treatment of severe infections due to Gram-positive bacteria, including staphylococci, enterococci, and streptococci. These antibiotics inhibit late stages in bacterial cell wall peptidoglycan synthesis? Their mode of action is unique and differs from that of the ~-lactams. Glycopeptides bind to peptidoglycan precursors that contain Dalanyl-D-alanine at their free carboxyl end and prevent, by steric hindrance, polymerization and cross-link of peptidoglycan (Figure 1). Enterococci constitute a normal component of the human intestinal flora but can provoke opportunistic infections in compromised patients. 2 These organisms have been implicated mostly in urinary tract infections, abdominal abscesses, and endocarditis episodes. Enterococcal infections cause significant therapeutic difficulties because of the high incidence of isolates multiply resistant to antimicrobial agents. 3 Recently, there have been a number of reports of the isolation of vancomycin-resistant enterococci from various parts of the world, including Europe and the USA. 4-7 The species involved include Enterococcus faecium, E. faecalis, E. avium, E. gallinarum, and E. casseliflavus. The resistant strains can be divided into three phenotypic classes, VANA, VANB, and VANC (Table 1), on the basis of the level of resistance to glycopeptides, cross-resistance between vancomycin and teicoplanin, and inducibility or constitutivity of resistance (Table 1). 8 VANA strains, mainly E. faecium, are inducibly resistant to high levels of vancomycin and teicoplanin. Resistance is inducible by glycopeptides only, either vancomycin or teicoplanin, and induced cells are cross-resistant to all glycopeptides. 9 There is no cross-resistance between glycopeptides and lipopeptide (daptomycin) or glycolipodepsipeptide (ramoplanin) antibiotics 4 resulting from differences in the mechanisms of action of the three groups of drugs. 1 VANB strains have inducible moderate levels of resistance to vancomycin and remain susceptible to teicoplanin? ° These strains of E. faecium and E. faecalis are less represented among clinical isolates in Europe but their incidence may be underestimated because this phenotype is difficult to detect by the disk-agar diffusion test and by automated techniques for antibiotic susceptibility determination. s E. gallinarum constitutes the VANC class; this bacterial species exhibits constitutive resistance to vancomycin