Products containing biocides: perceptions and realities

The mechanisms of action for chemical germicides and antibiotics for inactivating microorganisms are significantly different and methods for determining resistance by microorganisms to these agents are also different. Chemical germicides usually have multiple targets and the mechanisms for inactivation and resistance are not measured in absolute terms but rather in the rapidity with which they reduce levels of microorganisms. The term tolerance is much more suited for germicides than the term resistance. The mechanism of resistance to chemical germicides is often dependent on the concentration of the germicide. At high concentrations multiple cellular and metabolic targets are involved, and at low concentrations fewer cellular targets. In contrast antibiotics usually have a singular cellular or metabolic target and resistance implies the ability of the microorganism to grow in the presence of the antibiotic, and in a clinical sense, to initiate or continue infection in the presence of the antibiotic. When methods used to assess resistance to antibiotics are applied to chemical germicides, inappropriate interpretations can be made regarding the ability of microorganisms to develop resistance to antibiotics as a result of developing resistance to chemical germicides. The use of chemical germicides in health-care institutions and especially the home setting has increased in recent years. Although there may be an overuse of germicides in these settings the consequence is a cost issue and not one that involves the development of antibiotic resistant microorganisms.     

Structural insights for the substrate recognition mechanism of LL-diaminopimelate aminotransferase

The enzymes involved in the lysine biosynthetic pathway have long been considered to be attractive targets for novel antibiotics due to the absence of this pathway in humans. Recently, a novel pyridoxal 5'-phosphate (PLP) dependent enzyme called LL-diaminopimelate aminotransferase (LL-DAP-AT) was identified in the lysine biosynthetic pathway of plants and Chlamydiae. Understanding its function and substrate recognition mechanism would be an important initial step toward designing novel antibiotics targeting LL-DAP-AT. The crystal structures of LL-DAP-AT from Arabidopsis thaliana in complex with various substrates and analogues have been solved recently. These structures revealed how L-glutamate and LL-DAP are recognized by LL-DAP-AT without significant conformational changes in the enzyme's backbone structure. This review article summarizes the recent developments in the structural characterization and the inhibitor design of LL-DAP-AT from A. thaliana. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

头孢菌素C生物合成调控研究进展北大核心CSCD

头孢菌素C由丝状真菌顶头孢霉产生,属于β-内酰胺类抗生素。其经改造后的7-氨基头孢烷酸是头孢类抗生素的重要中间体。头孢类抗生素在国内外抗生素市场中占有巨大的份额,是临床上的主要抗感染药物。随着分子生物学的发展,头孢菌素C的生物合成途径已基本阐明。为提高头孢菌素C的产量和降低生产成本,越来越多的研究者开始关注其较为精细、复杂的调控机制。本文重点对头孢菌素C生物合成及其调控机制的最新进展进行了简述,希望为今后头孢菌素C生产菌株的菌种改造和传统产业的升级换代提供一定的借鉴。     

Genetics of Streptomyces rimosus, the oxytetracycline producer.

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.     

多氧霉素及其生物合成的研究进展

多氧霉素(Polyoxins)是高效广谱抗真菌核苷类抗生素,在农业上广泛用于防治植物真菌病害。本文综述了多氧霉素化学结构和理化性质,尤其是武汉大学组合生物合成与新药发现(教育部)重点实验室近年来在该抗生素生物合成基因簇的克隆、生物合成途径的阐明以及多氧霉素组合生物合成等多个方面的研究进展与成果,并对今后以多氧霉素为代表的核苷类抗生素的生物合成研究进行了展望。     

Effects of disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) and tetracyclines on drug resistant bacteria. Studies in vitro

An effect of Na2EDTA and tetracycline (oxytetracycline and doxycycline) resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa was tested. The strains were isolated from clinical specimens. The tests were performed in vitro by serial dilutions of the drugs in liquid medium. MIC for Na2EDTA, tetracyclines and a combination of Na2EDTA and tetracyclines was determined. It was shown that the combination of oxytetracycline or doxycycline with Na2EDTA caused changes in sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa resistant to these antibiotics. After an application of the mixture of various concentrations of tetracycline and Na2EDTA it was observed that, with the reduction of the effective Na2EDTA dose by about half, the lowest concentrations of tetracyclines inhibiting the growth of resistant bacteria were 2-64 times lower than MIC values of antibiotics without Na2EDTA.     

Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.     

Tigecycline is modified by the flavin-dependent monooxygenase TetX

The clinical use of tetracycline antibiotics has decreased due to the emergence of efflux and ribosomal protection-based resistance mechanisms. Currently in phase III clinical trials, the glycylcycline derivative tigecycline (GAR-936) containing a 9-tert-butylglycylamido group is part of a new generation of tetracycline antibiotics developed during the 1990s. Tigecycline displays a broad spectrum of antibacterial activity and circumvents the efflux and ribosomal protection resistance mechanisms. The TetX protein is a flavin-dependent monooxygenase that modifies first and second generation tetracyclines and requires NADPH, Mg(2+), and O(2) for activity. We report that tigecycline is a substrate for TetX and that bacterial strains containing the tet(X) gene are resistant to tigecycline. The resistance is due to the modification of tigecycline by TetX to form 11a-hydroxytigecycline, which we have shown has a weakened ability to inhibit protein translation compared with tigecycline. We have explored the basis of this decreased ability to block translation and found that hydroxylation occurs in the region of the molecule important for coordinating magnesium. 11a-Hydroxytigecycline forms a weaker complex with magnesium than tigecycline; the crystal structure of tetracycline in complex with the ribosome has shown that magnesium coordination is critical for binding tetracycline. Although tet(X) has not been isolated from any clinically resistant strains, our report demonstrates the first enzymatic resistance mechanism to tigecycline and provides an alert for the surveillance of resistant strains that may contain tet(X).     

Confirmation of oxytetracycline, tetracycline and chlortetracycline residues in milk by particle beam liquid chromatography/mass spectrometry.

A method using particle beam liquid chromatography/mass spectrometry was developed for the confirmation of oxytetracycline, tetracycline and chlortetracycline residues in bovine milk. This method is one of the first to apply particle beam technology to the confirmation of animal drug residues in food products for regulatory purposes. The milk is centrifuged, using molecular weight cut-off filters to remove components of 25,000 daltons and above from the milk. The filtrate is passed through a C-18 sample preparation cartridge which retains the tetracyclines. After the columns are washed with water, the tetracyclines are eluted with 0.1 M oxalic acid in methanol and concentrated. The compounds are separated on a Novapak C-18 column with a methanol-oxalic acid-acetonitrile mobile phase. Negative chemical ionization with selective ion monitoring is used to identify the tetracyclines. The procedure was used to confirm the presence of each tetracycline at 100 ng ml-1 in fortified and incurred milk samples.     

Genetics of Streptomyces rimosus, the Oxytetracycline Producer

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.     

Effects of tetracyclines on the production of extracellular proteins by members of the propionibacteriaceae

The effect of tetracyclines on the synthesis of proteins in Propionibacterium avidum and P. acnes was examined. Synthesis of an extracellular lipase by P. avidum was slightly more sensitive to inhibition by tetracycline than total (cellular and extracellular) protein synthesis. The effect of tetracycline and other analogues on the synthesis of secreted proteins was also examined in P. avidum and P. acnes by protein radiolabelling experiments. In all cases the synthesis of secreted proteins was only about 2-fold more sensitive to inhibition by tetracyclines than total protein synthesis. These results contrast with previously published findings in Escherichia coli which show that synthesis of secreted proteins is highly susceptible to inhibition by tetracycline. The implications of these findings in relation to inhibition of membrane bound ribosomes by tetracyclines are discussed.     

Experimental data on hygienic norms for antibiotics of the tetracycline group]

Toxicity of tetracyclines was studied experimentally on different species of laboratory animals. It was shown that tetracycline, oxytetracycline and chlortetracycline were close by their chemical structure and physico-chemical properties, as well as by the main toxicity parameters, i.e. acute toxicity, cumulative activity, skin-irritating and sensitizing effect. Under the conditions of subacute experiments the above 3 antibiotics induced evenly pronounced one direction changes in animals. The data obtained during the experiments provided recommendation of the level of 0.1 mg/m3 as the maximum allowable concentration (MAC) of oxytetracycline and chlortetracycline, i.e. the same level as the previously recommended for tetracycline.     

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment. To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators - including inorganic ions, antibiotics, and antibiotic resistance genes - were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 microg/L. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes - tet(M), tet(O), tet(Q), and tet(W) - were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

Decoding and engineering tetracycline biosynthesis

Tetracyclines have been important agents in combating infectious disease since their discovery in the mid-20th century. Following widespread use, tetracycline resistance mechanisms emerged and continue to create a need for new derivatives that are active against resistant bacterial strains. Semisynthesis has led to second and third generation tetracycline derivatives with enhanced antibiotic activity and pharmacological properties. Recent advancement in understanding of the tetracycline biosynthetic pathway may open the door to broaden the range of tetracycline derivatives and afford analogs that are difficult to access by synthetic chemistry.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

Molecular docking and enzymatic evaluation to identify selective inhibitors of aspartate semialdehyde dehydrogenase

Microbes that have gained resistance against antibiotics pose a major emerging threat to human health. New targets must be identified that will guide the development of new classes of antibiotics. The selective inhibition of key microbial enzymes that are responsible for the biosynthesis of essential metabolites can be an effective way to counter this growing threat. Aspartate semialdehyde dehydrogenases (ASADHs) produce an early branch point metabolite in a microbial biosynthetic pathway for essential amino acids and for quorum sensing molecules. In this study, molecular modeling and docking studies were performed to achieve two key objectives that are important for the identification of new selective inhibitors of ASADH. First, virtual screening of a small library of compounds was used to identify new core structures that could serve as potential inhibitors of the ASADHs. Compounds have been identified from diverse chemical classes that are predicted to bind to ASADH with high affinity. Next, molecular docking studies were used to prioritize analogs within each class for synthesis and testing against representative bacterial forms of ASADH from Streptococcus pneumoniae and Vibrio cholerae. These studies have led to new micromolar inhibitors of ASADH, demonstrating the utility of this molecular modeling and docking approach for the identification of new classes of potential enzyme inhibitors.     

Sensitivity of bifidobacteria to antibacterial drugs

Twenty one strains of bifidobacteria belonging to various species were studied with respect to their sensitivity to antibacterial drugs most widely used in medical practice. The strains were isolated from practically healthy persons and from persons in contact with antibiotics in their production. It was found that sensitivity of the strains was the highest with respect to penicillins and tetracyclines. With respect to kanamycin, monomycin and levomycetin the strains were resistant. Strain differences in resistance to separate antibacterial drugs were observed. Increased streptomycin and tetracycline resistance of the strains isolated from the persons being for a prolonged period in contact with these antibiotics in their production, was stated.     

Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK,p38, and nuclear factor-κB signaling pathways

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml) were added after treatment with LPS (10 μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1β was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course.     

Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Bacterial peptidoglycan is the cell wall component responsible for various biological activities. Its cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is biosynthesized by the first six enzymes of peptidoglycan synthetic pathways (Mur enzymes), which are all proved to be important targets for antibiotic screening. In our present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system, and the activities of Mur enzymes derived from CFPS system were validated by the synthesis of the final product UDP-N-acetylmuramyl pentapeptide. Then this in vitro reconstituted Mur biosynthetic pathway was used to screen a panel of specific antisense oligonucleotides for MurA and MurB. The selected oligonucleotides were proved to eliminate the expression of Mur enzymes, and thus inhibit the Mur biosynthetic pathway. The present work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.