Antibiotics Inhibit Protein Synthesis Research Articles

Inactivation of the ribosome assembly factor RimP causes streptomycin resistance and impairs motility in Salmonella.

The ribosome is the central hub for protein synthesis and the target of many antibiotics. Although the majority of ribosome-targeting antibiotics inhibit protein synthesis and are bacteriostatic, aminoglycosides promote protein mistranslation and are bactericidal. Understanding the resistance mechanisms of bacteria against aminoglycosides is not only vital for improving the efficacy of this critically important group of antibiotics but also crucial for studying the molecular basis of translational fidelity. In this work, we analyzed Salmonella mutants evolved in the presence of the aminoglycoside streptomycin (Str) and identified a novel gene rimP to be involved in Str resistance. RimP is a ribosome assembly factor critical for the maturation of the 30S small subunit that binds Str. Deficiency in RimP increases resistance against Str and facilitates the development of even higher resistance. Deleting rimP decreases mistranslation and cellular uptake of Str and further impairs flagellar motility. Our work thus highlights a previously unknown mechanism of aminoglycoside resistance via defective ribosome assembly.

Context-based sensing of orthosomycin antibiotics by the translating ribosome.

Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.

Comparison of protein synthesis inhibiting antibiotics versus cell wall inhibitor antibiotics for treatment of diabetic foot.

Diabetic foot infection is a form of soft tissue infection which rapidly involves the tissues of foot. It can affect all parts of foot but pressure areas of foot are commonly involved. Early diagnosis and treatment with proper antibiotic with or without surgical intervention are vital because of high morbidity. Objectives: To compare efficacy of protein synthesis-inhibiting antibiotics (clindamycin) versus cell wall inhibitor (imipenum) for treatment of diabetic foot infections as empirical therapy in term of clearance of infection and wound healing. Study Design: Randomized Clinical Trial. Setting: Surgical department POF Hospital. Period: January 2013 to January 2017. Material & Methods: Total of 94 patients of diabetic foot infection were included in the study through non-probability consecutive sampling. Divided into two groups each have 47 patients. In group A patients were given intravenous (i/v) imipenum while in group B intravenous (i/v) clindamycin was given. Pre and post treatment culture from wound was taken and healing observed in form of granulation tissue. Results: Group A patients (imipenum group) wound healing occurred in only 9 ( 19.1%) patients and in group B (clindamycin group) treatment was effective in 34 (72.3%) patients( P 0.001) Clearance of infection occurred in 31.91 %(15) in group A and 80.85%(38) in group B(P=0.001). Conclusion: Protein synthesis inhibitor antibiotics have shown increased efficacy for control of infection and healing as compared to cell wall inhibitor antibiotics.

Uneven host cell growth causes lysogenic virus induction in the Baltic Sea.

In the Baltic Sea redoxcline, lysogenic viruses infecting prokaryotes have rarely been detected using the commonly used inducing agent mitomycin C. However, it is well known that not all viruses are induceable by mitomycin C and growing evidence suggests that changes in trophic conditions may trigger the induction of lysogenic viruses. We hypothesized that using antibiotics to simulate a strong change in trophic conditions for antibiotica-resistant cells due to reduced competition for resources might lead to the induction of lysogenic viruses into the lytic cycle within these cells. This hypothesis was tested by incubating prokaryotes obtained throughout the Baltic Sea redoxcline in seawater with substantially reduced numbers of viruses. We used a mixture of the protein synthesis-inhibiting antibiotics streptomycin and erythromycin to induce the desired changes in trophic conditions for resistant cells and at the same time ensuring that no progeny viruses were formed in sensitive cells. No inducible lysogenic viruses could be detected in incubations amended with mitomycin C. Yet, the presence of streptomycin and erythromycin increased virus-induced mortality of prokaryotes by 56–930% compared to controls, resulting in the induction of lysogenic viruses equivalent to 2–14% of in situ prokaryotic abundance. The results indicate the existence of a previously unrecognized induction mechanism for lysogenic viruses in the Baltic Sea redoxcline, as the mode of action distinctly differs between the used antibiotics (no virus production within affected cells) and mitomycin C (lysogenic viruses are produced within affected cells). Obtaining accurate experimental data on levels of lysogeny in prokaryotic host cells remains challenging, as relying on mitomycin C alone may severely underestimate lysogeny.

The Anti-Staphylococcal Potential of Ethanolic Polish Propolis Extracts.

The principal objective of this study was to determine the anti-staphylococcal potential of ethanol extracts of propolis (EEPs). A total of 20 samples of propolis collected from apiaries located in different regions of Poland were used in the study. The two-fold broth microdilution method revealed some important differences in the antimicrobial activity of investigated EEPs. Up to the concentration of 4096 µg/mL no activity was observed against Gram-negative bacteria (E. coli and P. aeruginosa). Staphylococci exhibited much higher susceptibility. The highest efficiency observed for EEP12 and EEP20 (MIC values ranged between 32 and 256 µg/mL). However, the achievement of bactericidal effect usually required higher concentrations. In the case of clinical isolates of S. aureus MBC values for EEP12 and EEP20 ranged from 512 to 1024 µg/mL. The HPLC analysis revealed that these two products contained a higher concentration of flavonoids (flavonols, flavones, and flavanones) compared to other investigated EEPs. In checkerboard test, a synergistic anti-staphylococcal effect was observed for the action of EEP20 in combination with amikacin, kanamycin, gentamycin, tetracycline, and fusidic acid (all these antibiotics inhibit protein synthesis). Moreover, the investigated EEPs effectively eradicated staphylococcal biofilm. The obtained results clearly confirm the high anti-staphylococcal potential of propolis harvested in Polish apiaries.

Rapid Determination of Resistance to Antibiotic Inhibitors of Protein Synthesis in Staphylococcus aureus Through In Situ Evaluation of DNase Activity.

A rapid assay was designed for the detection of resistant strains of Staphylococcus aureus to antibiotic inhibitors of protein synthesis. The assay was based on the fact that a brief cell wall digestion with lysostaphin resulted in fragmentation of the chromosomal DNA by releasing the characteristic DNase stored in the cell wall. DNase activity was ascertained by visualization of the DNA fragments released from the isolated nucleoids. Lysostaphin-released DNase activity was found to be influenced by ribosomal protein synthesis. Inhibition of protein synthesis resulted in the prevention of lysostaphin-DNase induced DNA fragmentation when susceptible clinical strains were incubated with erythromycin, azithromycin, or doxycycline for 2 hr before enzymatic treatment. However, in nonsusceptible strains where protein synthesis was unsuccessfully inhibited, this suppression of lysostaphin-DNase was not, or only very slightly, evident. This assay was highly efficient, identifying resistance to erythromycin and azithromycin with 88-90.9% sensitivity and 100% specificity and with 100% sensitivity and specificity to gentamicin and doxycycline, within a 2 hr and 45 min period.

Progress of research on the toxicology of antibiotic pollution in aquatic organisms

Antibiotics are widely used to improve human and animal health and treat infections. Antibiotics are often used in livestock farms and fisheries to prevent diseases and promote growth. Recently, there has been increasing interest in the presence of antibiotics in aquatic environments. Low levels of antibiotic components are frequently detected in surface water, seawater, groundwater, and even drinking water. Antibiotics are consistently and continually discharged into the natural environment as parent molecules or metabolites, which are usually soluble and bioactive, and this results in a pseudo and persistent pollution. The effects of environmental antibiotic toxicity on non-target organisms, especially aquatic organisms, have become an increasing concern. Although antibiotics have been detected worldwide, their ecological and developmental effects have been poorly investigated, particularly in non-target organisms. This review describes the toxicity and underlying mechanism of antibiotic contamination in aquatic organisms, including the effects on vertebrate development. A considerable number of antibiotic effects on aquatic organisms have been investigated using acute toxicity assays, but only very little is known about the long-term effects. Aquatic photosynthetic autotrophs, such as Pseudokirchneriella subcapitata, Anabaena flos-aquae, and Lemna minor, were previously used for antibiotic toxicity tests because of low cost, simple operation, and high sensitivity. Certain antibiotics show a different degree of potency in algal toxicity tests on the basis of different test algae. Antibiotics inhibit protein synthesis, chloroplast development, and photosynthesis, ultimately leading to growth inhibition; some organisms exhibit growth stimulation at certain antibiotic concentrations. Daphnia magna and other aquatic invertebrates have also been used for checking the toxicity priority of antibiotics. When investigating the acute effect of antibiotics (e.g., growth inhibition), concentrations in standard laboratory organisms are usually about two or three orders of magnitude higher than the maximal concentrations in the aquatic environment, resulting in the underestimation of antibiotic hazards. Vertebrate organisms show a promising potential for chronic toxicity and potentially subtle effects of antibiotics, particularly on biochemical processes and molecular targets. The adverse developmental effects of macrolides, tetracyclines, sulfonamides, quinolones, and other antibiotic groups have been evaluated in aquatic vertebrates such as Danio rerio and Xenopus tropicalis. In acute toxicity tests, low levels of antibiotics have systematic teratogenic effects on fish. The effects of antibiotics on oxidative stress enzymes and cytochrome P450 have been investigated. Cytotoxicity, neurotoxicity, and genotoxicity have been observed for certain antibiotic amounts. However, there are no firm conclusions regarding the chronic toxicity of antibiotics at environmentally relevant levels because of the lack of long-term exposure studies. Herein, future perspectives and challenges of antibiotic toxicology were discussed. Researchers should pay more attention to the following points: chronic toxicity and potentially subtle effects of environmentally relevant antibiotics on vertebrates; effects of toxicity on biochemical processes and mode of action; combined toxicity of antibiotics and other antibiotics, metabolites, and heavy metals; and environmental factors such as temperature and pH.

Simple and Fast Detection of Resistance to Antibiotic Inhibitors of Protein Synthesis in Gram-Negative Pathogens Through Evaluation of Mitomycin C-Induced Cell Elongation.

Increasing the resistance of Gram-negative pathogens to antibiotics that inhibit protein synthesis is of great concern. In life-threatening situations, an early detection of antibiotic resistance may improve patient outcome. A rapid assay for the identification of antibiotic resistance to gentamicin, tobramycin, and tigecycline has been designed and tested in clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa, and the Enterobacteriaceae Escherichia coli and Klebsiella pneumoniae. Exponentially growing cultures were incubated with 0.5 mg/L mitomycin C (MMC) for 2 hr (10 mg/L for A. baumannii), which induced significant cell enlargement as visualized under the microscope. Addition of the appropriate antibiotic dose 15 min before the addition of MMC prevented elongation when the strain was susceptible to the antibiotic, thereby inhibiting protein synthesis. Cell enlargement was not precluded in the antibiotic resistant strains, where protein synthesis had not been successfully inhibited. In comparison with the standard dilution-based antibiogram, the sensitivity of the assay was 100% and the specificity ranged between 96.0% and 100%. Results were obtained after 2 hr and 45 min from exponentially growing cultures. The procedure is easy, reliable, and demonstrates the suitability of the evaluation of simple morphological changes, which are protein synthesis dependent, for the rapid detection of antibiotic resistance.

Rapid Assessment of Resistance to Antibiotic Inhibitors of Protein Synthesis in the Gram-Positive Pathogens, Enterococcus faecalis and Streptococcus pneumoniae, Based on Evaluation of the Lytic Response.

A novel assay for rapid determination of resistance to antibiotic inhibitors of protein synthesis was developed for the gram-positive pathogens, Enterococcus faecalis and Streptococcus pneumoniae. To this purpose, a lytic response was obtained by a brief incubation with lysozyme or a mixture of lysozyme, Triton X-100, and EDTA for E. faecalis (n = 82) and S. pneumoniae (n = 51), respectively. Lysis was quantified by visualizing the released nucleoids. Antibiotic-susceptible bacteria treated with Clinical and Laboratory Standards Institute (CLSI) breakpoint doses of erythromycin, azithromycin, or doxycycline that inhibited protein synthesis demonstrated a large reduction of lysed cells with respect to the control, that is, without antibiotics. However, cell lysis prevention was much lower in nonsusceptible strains, with unsuccessful inhibition of protein synthesis. ROC analysis showed that a reduction value of ≥35.6% and ≥40.4% discriminates susceptible and nonsusceptible strains for erythromycin and for doxycycline, respectively, in E. faecalis, whereas ≥20.0% is adequate for both macrolides and doxycycline in S. pneumoniae. Resistant stains were identified in 90-120 min with sensitivity and specificity between 91.7% and 100%. This is a proof of concept that evaluation of the lytic response may be a rapid and efficient test for determination of resistance to antibiotic inhibitors of protein synthesis.

Evidence That Antibiotics Bind to Human Mitochondrial Ribosomal RNA Has Implications for Aminoglycoside Toxicity

Aminoglycosides are a well known antibiotic family used to treat bacterial infections in humans and animals, but which can be toxic. By binding to the decoding site of helix44 of the small subunit RNA of the bacterial ribosome, the aminoglycoside antibiotics inhibit protein synthesis, cause misreading, or obstruct peptidyl-tRNA translocation. Although aminoglycosides bind helix69 of the bacterial large subunit RNA as well, little is known about their interaction with the homologous human helix69. To probe the role this binding event plays in toxicity, changes to thermal stability, base stacking, and conformation upon aminoglycoside binding to the human cytoplasmic helix69 were compared with those of the human mitochondrial and Escherichia coli helix69. Surprisingly, binding of gentamicin and kanamycin A to the chemically synthesized terminal hairpins of the human cytoplasmic, human mitochondrial, and E. coli helix69 revealed similar dissociation constants (1.3-1.7 and 4.0-5.4 μM, respectively). In addition, aminoglycoside binding enhanced conformational stability of the human mitochondrial helix69 by increasing base stacking. Proton one-dimensional and two-dimensional NMR suggested significant and specific conformational changes of human mitochondrial and E. coli helix69 upon aminoglycoside binding, as compared with human cytoplasmic helix69. The conformational changes and similar aminoglycoside binding affinities observed for human mitochondrial helix69 and E. coli helix69, as well as the increase in structural stability shown for the former, suggest that this binding event is important to understanding aminoglycoside toxicity.

Detection of tetracycline resistance genes by PCR methods.

Rapid, accurate, and sensitive determination of antibiotic resistance profiles of various human and animal pathogens becomes a vital prerequisite for successful therapeutic intervention in the face of the increased occurrences of drug-resistant bacterial infections. The current methods, which are dependent on cultivation of pathogens and phenotypic expression of antibiotic resistance, usually require excessive time, special microbiological equipment, and qualified personnel. However, even with all these requisites, for example, no bacteria can be grown from more than 80% of all clinical samples sent to clinical microbiology laboratories. Besides the cultivation limitations, the cultivation-based determination of an antibiotic resistance profile lacks the genotypic information, which is essential for understanding the epidemiology and routes of transmission of antibiotic resistance genes. These genes often reside on mobile genetic elements and can move freely between commensal and pathogenic microbiota, occurring even between taxonomically distant clinical and environmental microbiota. Therefore, development of genotyping methods for detection of antibiotic resistance genes is highly desirable for fast, accurate, and sensitive detection of antibiotic resistance genes in a broad range of pathogenic and commensal bacteria in both clinical and environmental samples. As a model for our studies we have chosen the genes conferring resistance to tetracyclines. Tetracyclines belong to a family of broad-spectrum antibiotics that include tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, and a number of other semisynthetic derivatives. These antibiotics inhibit protein synthesis in Gram-positive and Gram-negative bacteria by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit. The antibiotics of this group were introduced in the late 1950s and since then have been widely used in clinical and veterinary medicine, as well as for prophylaxis and growth promotion in food animals. Because of the possible misuse and overuse of these drugs, resistance to this class of antibiotics is widespread among many clinical isolates, thus limiting the utility of tetracyclines in treating infections. Despite this shortcoming, antibiotics of this class still remain in the active arsenal for dermatologists to treat skin infections such as acne and rosacea.

Isolation and phenotypic characteristics of the Escherichia coli butanol-tolerant mutants

Resistance to butanol is a key factor affecting microbial ability to produce economically profitable amounts of butanol. In this study, an Escherichia coli strain capable of growth in the presence of 1.5% butanol was isolated. The mutant MG1655 ButR was characterized by increased resistance to ethanol, isopropanol, and bivalent ions but exhibited supersensitivity to osmotic shock. Compared to the wild type strain, the butanol-tolerant mutant was more sensitive to antibiotics inhibiting protein synthesis but was more resistant to membrane-penetrating antibiotics, such as surfactin. Increased content of unsaturated fatty acids was found in the membranes of butanol-tolerant mutants. It was revealed that overexpression of the genes encoding cold-shock proteins decreased butanol tolerance of both mutant and the wild-type strain. It was concluded that butanol tolerance was associated with multiple rearrangements of the cell genetic system, rather than with single mutations.

SigB-Dependent Tolerance to Protein Synthesis-Inhibiting Antibiotics in Listeria monocytogenes EGDe

The alternative sigma factor SigB in food-borne pathogen Listeria monocytogenes was determined in this study to be required for tolerance to protein synthesis-inhibiting antibiotics. The minimum inhibitory concentrations of tetracycline HCl and gentamicin sulphate against EGDeΔsigB were two- and fourfold less than those for EGDe, respectively. The ability of EGDeΔsigB to overcome the growth arrest caused by erythromycin and rifampin was also weaker than that of EGDe. The transcription analysis of four genetic loci (known to be induced by rifampin in Bacillus subtili) kat, fri, ropB and rsbU in EGDe and EGDeΔsigB in the absence or presence of rifampin revealed that: (1) expression of kat and fri genes is σ (B) dependent, but only the former is inducible by rifampin stress; (2) the transcriptional level of rpoB gene was stable under all the experimental conditions, while that of rsbU in EGDeΔsigB was remarkably higher in the absence of rifampin and significantly increased in EGDe but reduced in EGDeΔsigB after rifampin application, when compared to those in EGDe and EGDeΔsigB control without antibiotic, respectively. These results suggest that complex physiological reactions to tolerance of the antibiotic stress are variably regulated in bacteria, and in contrast to B. subtilis, rsbU in EGDeΔsigB may compensate for the σ (B)-dependent genes that are necessary for tolerance to rifampin stress and therefore plays a role in overcoming the antibiotic-triggered growth arrest.

Inhibitors of the Large Ribosomal Subunit from Haloarcula marismortui

AbstractThe crystal structures that have been obtained for 23 different inhibitors bound to the large ribosomal subunit from Haloarcula marismortui are reviewed here. These structures provide important insights into how anti‐ribosomal antibiotics inhibit protein synthesis, how species specificity arises, and the relationship between ribosomal mutations and antibiotic resistance. These structural studies also provide compelling evidence that the conformation of the peptidyl transferase center of the large ribosomal subunit is intrinsically variable, and that conformational equilibria play a role in determining its functional properties.

Limitations of antibiotic options for invasive infections caused by methicillin-resistant Staphylococcus aureus: is combination therapy the answer?

Invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA), particularly those involving persistent bacteraemia, necrotizing pneumonia, osteomyelitis and other deep-seated sites of infections, are associated with high mortality and are often difficult to treat. The response to treatment of severe MRSA infection with currently available antibiotics active against MRSA is often unsatisfactory, leading some physicians to resort to combination antibiotic therapy. Now, with the emergence of community-associated MRSA (CA-MRSA) clones that display enhanced virulence potentially related to up-regulated toxin production, the use of adjuvant protein synthesis-inhibiting antibiotics to reduce toxin production also has been advocated by some experts. In this review, we discuss the limitations of antibiotics currently available for the treatment of serious invasive MRSA infections and review the existing literature that examines the potential role of combination therapy in these infections.

Antibiotic inhibitors of protein synthesis on the ribosome

Many types of chemically diverse antibiotics inhibit bacterial growth by binding to the ribosome and blocking protein synthesis. The large majority of these antibiotics have a distinct target site on either the 30S or the 50S ribosomal subunit. Thus, these antibiotics are generally specific inhibitors of steps in protein synthesis that are associated with either the 30S subunit (such as mRNA decoding) or the 50S subunit (such as peptide bond formation or controlled GTP hydrolysis). An important exception to this rule is the cyclic peptide drug, capreomycin, which is currently used to treat strains of Mycobacterium tuberculosis that have become resistant to first‐line drug therapy. Capreomycin and related drugs function by binding across the ribosomal subunit interface to simultaneously interact with both the 30S and the 50S, where they block the translocation of tRNA along the messenger RNA. Resistance to many ribosome‐targeting drugs is often conferred by addition of one or more methyl groups to specific nucleotides in the rRNA, and thereby sterically hindering the binding of the antibiotic to its target. Resistance to capreomycin has been analyzed in clinical strains of M. tuberculosis, and the mechanism is exceptional in that it is conferred by loss of natural methylations on the rRNA, rather than by gaining extra methylations.

Linezolid and tiamulin cross-resistance in Staphylococcus aureus mediated by point mutations in the peptidyl transferase center.

Oxazolidinone and pleuromutilin antibiotics are currently used in the treatment of staphylococcal infections. Although both antibiotics inhibit protein synthesis and have overlapping binding regions on 23S rRNA, the potential for cross-resistance between the two classes through target site mutations has not been thoroughly examined. Mutants of Staphylococcus aureus resistant to linezolid were selected and found to exhibit cross-resistance to tiamulin, a member of the pleuromutilin class of antibiotics. However, resistance was unidirectional because mutants of S. aureus selected for resistance to tiamulin did not exhibit cross-resistance to linezolid. This contrasts with the recently described PhLOPS(A) phenotype, which confers resistance to both oxazolidinones and pleuromutilins. The genotypes responsible for the phenotypes we observed were examined. Selection with tiamulin resulted in recovery of mutants with changes in the single-copy rplC gene (Gly155Arg, Ser158Leu, or Arg149Ser), whereas selection with linezolid led to recovery of mutants with changes (G2576U in 23S rRNA) in all five copies of the multicopy operon rrn. In contrast, cross-resistance to linezolid was exhibited by tiamulin-resistant mutants generated in a single-copy rrn knockout strains of Escherichia coli, illustrating that the copy number of 23S rRNA is the limiting factor in the selection of 23S rRNA tiamulin-resistant mutants. The interactions of linezolid and tiamulin with the ribosome were modeled to seek explanations for resistance to both classes in the 23S rRNA mutants and the lack of cross-resistance between tiamulin and linezolid following mutation in rplC.

Activity of Garenoxacin against Macrolide-Susceptible and -Resistant Mycoplasma pneumoniae

In the treatment of Mycoplasma pneumoniae infection, erythromycin and clarithromycin, 14-membered macrolides, and azithromycin, a 15-membered macrolide, are usually considered to be the first-choice drugs (4, 11). Macrolide antibiotics inhibit protein synthesis by binding to domain II and/or domain V of 23S rRNA (1, 13). We found that ca. 20% of M. pneumoniae strains isolated from patients in Japan from 2000 to 2003 were macrolide resistant (6, 9). Lucier et al. (5) and Okazaki et al. (7) found that an A-to-G transition or an A-to-C transversion at position 2063 (corresponding to position 2058 in Escherichia coli) or 2064 of the 23S rRNA gene results in a high level of resistance to macrolide antibiotics. Given this background, it was considered that an alternative to macrolides in the treatment of pneumonia caused by M. pneumoniae is needed. Garenoxacin, a des-F(6)-quinolone that exhibits excellent activity against respiratory pathogens such as Streptococcus pneumoniae and Chlamydophila pneumoniae, is under development for both oral and parenteral administration (10). In the present study, we examined the in vitro activity of garenoxacin against M. pneumoniae isolates, including macrolide-resistant strains, and compared it with those of other antibiotics. The following agents were employed for MIC determinations: garenoxacin (Toyama Chemical Co., Ltd., Tokyo, Japan), gatifloxacin (Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan), levofloxacin and clarithromycin (LKT Laboratories, Inc., Saint Paul, MN), and minocycline (Lederle-Japan Ltd., Tokyo, Japan). The purity of each of these agents was above 99.8%. The following isolates of M. pneumoniae were tested: three macrolide-susceptible reference strains, FH, Mac, and M129, and four macrolide-resistant clinical isolates, 1 and 6 (isolates carrying the A-to-G mutation at position 2063 in the 23S rRNA gene for which the MIC of clarithromycin is 32 μg/ml) and 2 and 4 (isolates carrying the A-to-G mutation at position 2064 in the 23S rRNA gene for which the MIC of clarithromycin is 8 μg/ml). The PCR amplification and sequencing of domain II of the 23S rRNA genes were performed by a method reported previously (6). A broth microdilution method was used to determine the MICs. Serial twofold dilutions of antibacterial agents prepared in PPLO broth (Difco Inc., Detroit, MI) containing 104 to 105 CFU of M. pneumoniae/ml were put into 96-well microplates. The microplates were sealed with adhesive sheets and incubated at 37°C for 4 to 8 days. The MIC was defined as the lowest concentration of a drug at which the metabolism of the organism was inhibited, as evidenced by a lack of color change in the medium at the time when the drug-free control first showed a color change. The MICs of garenoxacin for the M. pneumoniae isolates, including the macrolide-resistant isolates, were 0.016 to 0.031 μg/ml, two- to fourfold lower than those of gatifloxacin and 8- to 32-fold lower than those of levofloxacin and minocycline (Table ​(Table1).1). The results for macrolide-susceptible strains such as M. pneumoniae FH, a reference strain, accorded with those given in previous reports (2, 8, 12). TABLE 1. Activities of various antibiotics against M. pneumoniae strains The newer fluoroquinolones such as gatifloxacin and moxifloxacin exhibit potent in vitro activity against a broad spectrum of organisms, including M. pneumoniae (3). Garenoxacin showed more-potent in vitro activity against M. pneumoniae than gatifloxacin and has attracted interest as a potential therapy for community-acquired pneumonia. These data indicate that garenoxacin may be a viable alternative to macrolides, such as clarithromycin, in the treatment of pneumonia caused by M. pneumoniae, including macrolide-resistant strains.

Mode of action of microbial bioactive metabolites

Pathogenic microorganisms can be suppressed by cell wall destruction. Biosynthesis of peptidoglycans forming bacterial cell wall is interrupted by glycopeptides which inhibit polymerization of a disaccharide formed by N-acetylglucosamine and N-acetylmuramic acid, beta-lactams and their derivatives inhibit peptidoglycan cross-linking. Antibiotics inhibiting protein synthesis bind to different sites on the rRNA and interfere with the formation of the polypeptide chain. Tumor cells resistant to chemotherapeutic drugs overproduce proteins transporting the drugs out of cells; these proteins eliminate substances which inhibit transcription of transport proteins. Some antitumor drugs (anthracyclines, fluoroquinolones, acridines etc.) act at topoisomerases which irreversibly bind to DNA and inhibit DNA synthesis. Immunosuppressants affect various components of the immune system such as T-helper, T-effector cell function, antigen presentation and B-cell function. Antiparasitics--avermectins--bind to a receptor of this Gab-gated chlorine channel in the nerve fiber of nematodes and anthropodes, increasing the permeability of the membrane for chloride ions; the increased transport of chloride ions into the cell causes the death of the parasite. Ionophores dissolve in phospholipid bilayers and enormously increase their ionic permeability. Respiration inhibitors block the transport of electrons at several places of the respiratory chain. Rifamycin binds to the beta subunit of bacterial RNA polymerase, thereby blocking mRNA synthesis. Antiviral compounds inhibit the transcription of DNA by several mechnisms or by inhibition of viral entry into host cells.

Structure-based drug design meets the ribosome

The high-resolution structures of the bacterial ribosomal subunits and those of their complexes with antibiotics have advanced significantly our understanding of small-molecule interactions with RNA. The wealth of RNA structural data generated by these structures has allowed computational chemists to employ a drug discovery paradigm focused on RNA-based targets. The structures also show how target-based resistance affects antibiotics acting at the level of the ribosome. Not only are the sites pinpointed where different classes of antibiotics inhibit protein synthesis, but their orientations, relative dispositions, and unique mechanisms of action are also revealed at the atomic level. Both the 30S and the 50S ribosomal subunits have been shown to be “targets of targets”, offering several adjacent, functionally relevant binding pockets for antibiotics. It is the detailed knowledge of these validated locations, or ribofunctional loci, plus the mapping of the resistance hot-spots that allow the rational design of next-generation antibacterials. When the structural information is combined with a data-driven computational toolkit able to describe and predict molecular properties appropriate for bacterial cell penetration and drug-likeness, a structure-based drug design approach for novel antibacterials shows great promise.