Effect of Macrolide Antibiotics on Macrophage Functions

Macrolide antibiotics have a variety of actions other than antimicrobial activities. Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effects of macrolide antibiotics on macrophage functions. For the macrophage, we used the mouse macrophage cell line J774.1. The following effects of macrolide antibiotics on macrophage functions were evaluated: the effect of macrolide antibiotics on macrophage growth; the phagocytosis of beads; cytocidal activity against Candida albicans; and chemotaxis to lipopolysaccharide (LPS). Macrolide antibiotics except for azithromycin significantly stimulated the growth of the macrophage. In addition, pretreatment with macrolide antibiotics except for roxithromycin significantly stimulated the macrophage phagocytosis of beads, macrophage chemotaxis to LPS, and macrophage cytocidal activity against Candida albicans. These results suggest that macrolide antibiotics stimulate macrophage functions.     

Influence of macrolide maintenance therapy and bacterial colonisation on exacerbation frequency and progression of COPD (COLUMBUS): Study protocol for a randomised controlled trial

ABSTRACT: BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by progressive development of airflow limitation that is poorly reversible. Because of a poor understanding of COPD pathogenesis, treatment is mostly symptomatic and new therapeutic strategies are limited. There is a direct relationship between the severity of the disease and the intensity of the inflammatory response. Besides smoking, one of the hypotheses for the persistent airway inflammation is the presence of recurrent infections. Macrolide antibiotics have bacteriostatic as well as anti-inflammatory properties in patients with cystic fibrosis and other inflammatory pulmonary diseases. There is consistent evidence that macrolide therapy reduces infectious exacerbations, decreases the requirement for additional antibiotics and improves nutritional measures. Because of these positive effects we hypothesised that maintenance macrolide therapy may also have beneficial effects in patients with COPD who have recurrent exacerbations. The effects on development of bacterial resistance to macrolides due to this long-term treatment are unknown. Until now, studies investigating macrolide therapy in COPD are limited. The objective of this study is to assess whether maintenance treatment with macrolide antibiotics in COPD patients with three or more exacerbations in the previous year decreases the exacerbation rate in the year of treatment and to establish microbial resistance due to the long-term treatment. Methods/design The study is set up as a prospective randomised double-blind placebo-controlled single-centre trial. A total of 92 patients with COPD who have had at least three exacerbations of COPD in the previous year will be included. Subjects will be randomised to receive either azithromycin 500 mg three times a week or placebo. Our primary endpoint is the reduction in the number of exacerbations of COPD in the year of treatment. DISCUSSION: We investigate whether long-term therapy with macrolide antibiotics can prevent exacerbations in patients with COPD. Additionally, our study aims to assess the effect of long-term use of macrolide on the development of antimicrobial resistance and on inflammatory parameters related to COPD. We believe this study will provide more data on the effects of macrolide treatment in patients in COPD and will add more knowledge on its working mechanisms. Trial registration www.clinicaltrials.gov NCT00985244.     

大环内酯类抗生素微生物降解的研究进展

大环内酯类抗生素是一类以大环内酯为母核的广谱抗生素。近些年,由于人们对其不规范的生产和使用,抗生素污染成为了重要的环境问题。大量研究表明,微生物降解是现阶段处理抗生素污染的最理想方法。为进一步推动大环内酯类抗生素生物降解的研究,文中概述了大环内酯类抗生素的环境污染现状、微生物降解菌株、降解酶、降解途径和降解大环内酯类抗生素的微生物处理方法,并对大环内酯类抗生素生物降解亟待解决的瓶颈问题进行了讨论,以期为微生物降解后续研究提供参考。     

酵母中甾醇定向存储转运及理性强化技术研究进展

大环内酯类抗生素是一类以大环内酯为母核的广谱抗生素。近些年,由于人们对其不规范的生产和使用,抗生素污染成为了重要的环境问题。大量研究表明微生物降解是现阶段处理抗生素污染的最理想方法。为进一步推动大环内酯类抗生素生物降解的研究,文中概述了大环内酯类抗生素的环境污染现状、微生物降解菌株、降解酶、降解途径和降解大环内酯类抗生素的微生物处理方法,并对大环内酯类抗生素生物降解亟待解决的瓶颈问题进行了讨论,以期为微生物降解后续研究提供参考。     

Adjuvant effect of a 14-member macrolide antibiotic on DNA vaccine

Macrolide antibiotics have unique immunomodulatory actions apart from their antimicrobial properties. We examined the effect of erythromycin (EM), a 14-member macrolide, on the immune response to a DNA vaccine that induces a T-helper-1 (Th1)-biased immune response through a Th1-promoting adjuvant effect of unmethylated CpG motifs within plasmid DNA. EM enhanced Th1 responses in plasmid DNA-immunized mice as measured by antigen-specific IgG2a antibody production, interferon-gamma production by antigen-specific CD4(+) T cells, and cytotoxic T lymphocyte responses. EM augmented the accessory cell activity of unmethylated CpG DNA-stimulated antigen-presenting cells (APCs), suggesting that EM enhances Th1 responses to a DNA vaccine, possibly through augmentation of accessory cell activity of APCs stimulated with CpG motifs within plasmid DNA.     

Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.     

Short peptides conferring resistance to macrolide antibiotics

Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases.     

Suppressive activity of macrolide antibiotics on nitric oxide production by lipopolysaccharide stimulation in mice

BACKGROUND: Low-dose and long-term administration of macrolide antibiotics into patients with chronic airway inflammatory diseases could favorably modify their clinical conditions. However, the therapeutic mode of action of macrolides is not well understood. Free oxygen radicals, including nitric oxide (NO), are well recognized as the important final effector molecules in the development and the maintenance of inflammatory diseases. PURPOSE: The influence of macrolide antibiotics on NO generation was examined in vivo. METHODS: Male ICR mice, 5 weeks of age, were orally administered with either roxithromycin, clarithromycin, azithromycin or josamycin once a day for 2-4 weeks. The mice were then injected intraperitoneally with 5.0 mg/kg lipopolysaccharide (LPS) and the plasma NO level was examined 6 h later. RESULTS: Although pre-treatment of mice with macrolide antibiotics for 2 weeks scarcely affected NO generation by LPS injection, the administration of macrolide antibiotics, except for josamycin, for 4 weeks significantly inhibited LPS-induced NO generation. The data in the present study also showed that pre-treatment of mice with macrolide antibiotics for 4 weeks significantly suppresses not only production of pro-inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, but also inducible nitric oxide synthase mRNA expressions, which are enhanced by LPS injection. CONCLUSION: These results strongly suggest that suppressive activity of macrolide antibiotics on NO generation in response to LPS stimulation in vivo may, in part, account for the clinical efficacy of macrolides on chronic inflammatory diseases.     

Lung inflammatory pattern and antibiotic treatment in pneumonia

Background

In community-acquired pneumonia host inflammatory response against the causative microorganism is necessary for infection resolution. However an excessive response can have deleterious effects. In addition to antimicrobial effects, macrolide antibiotics are known to possess immunomodulatory properties.We aimed to evaluate inflammatory cytokine profiles – both locally (bronchoalveolar lavage) and systemically (blood) – in community-acquired pneumonia admitted patients after at least 72 hours of antibiotic treatment (with and without macrolide containing regimens) and requiring bronchoscopic examination for inadequate response due to infection progression and/or lack of clinical stability.

Methods

A prospective study was performed on 52 admitted patients who developed an inadequate response after 72 hours of antibiotic treatment - non-responders community-acquired pneumonia - (blood and bronchoalveolar lavage), and two control groups: 1) community-acquired pneumonia control (blood) and 2) non-infection control (blood and bronchoalveolar lavage). Cytokine profiles (interleukin (IL)-6, IL-8, IL-10), tumour necrosis factor α and clinical outcomes were assessed.

Results

Non–responders patients treated with macrolide containing regimens showed significantly lower levels of IL-6 and TNF-α in bronchoalveolar lavage fluid and lower IL-8 and IL-10 in blood than those patients treated with non-macrolide regimens. Clinical outcomes showed that patients treated with macrolide regimens required fewer days to reach clinical stability (p < 0.01) and shorter hospitalization periods (p < 0.01).

Conclusions

After 72 hours of antibiotic effect, patients who received macrolide containing regimens exhibited lower inflammatory cytokine levels in pulmonary and systemic compartments along with faster stabilization of infectious parameters.     

Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages.

Macrolide antibiotics have unique immunomodulatory actions apart from antimicrobial properties. We studied the effects of macrolides on IgG immune complex (IgG-ICx)-induced lung injury in rats in vivo and in vitro. Intrapulmonary deposition of IgG-ICx produced a time-dependent increase in the concentration of NO in exhaled air. There were corresponding increases in the number of neutrophils accumulated into alveolar spaces, and lung wet-to-dry weight ratio. All of these changes were inhibited by pretreatment with erythromycin or josamycin, but not by amoxicillin or cephaclor. Incubation of cultured pulmonary alveolar macrophages caused up-regulation of NO production and expression of inducible NO synthase mRNA, an effect that was dose dependently inhibited by erythromycin, roxithromycin, or josamycin. The macrolides also reduced IgG-ICx-induced release of IL-1beta and TNF-alpha, but did not alter the release of NO induced by exogenously added IL-1beta and TNF-alpha. These results suggest that macrolide antibiotics specifically inhibit immune complex-induced lung injury presumably by inhibiting cytokine release and the resultant down-regulation of inducible NO synthase gene expression and NO production by rat pulmonary alveolar macrophages.     

Pidotimod: the state of art

Despite the use of antibiotics and vaccines, the frequency of respiratory tract infections is still high and these infections interest a wide range of patients, from children to aged people, including in particular these extreme categories because of the deficiency of their immune system, due to immaturity in the former case and to “immunosenescence” in the latter. For that reason immunostimulant drugs are getting more important to prevent and to attenuate infections. Pidotimod (3-L-pyroglutamyl-L-thiazolidine-4carboxylic acid) is a synthetic dipeptide with immunomodulatory properties. We reviewed studies conducted on different categories of patients, with particular attention on children and senile patients suffering from recurrent respiratory tract infections, associated, or not, with asthma or COPD. The outcomes considered are both clinical and laboratory parameters. The common end-point of these studies is that Pidotimod has an immunomodulatory activity which is able both to improve the clinical conditions of patients and to enhance and stimulate their immunity cells (lymphocytes but not only) functions acting on adaptive and innate immunity. Pidotimod is also able to increase the concentration of salivary IgA directed against bacteria; furthermore, it can modulate airway epithelial cells functions up-regulating the expression of toll-like receptors and acting on adhesion molecules. According to studies conducted on patients with atopic asthma, it seems that Pidotimod could affect T-lymphocytes balance with a possible addictional anti-allergic activity. Furthermore, it has been demonstrated an improvement of FEV1 and PEF in asthmatic patients treated with Pidotimod. Main clinical outcomes are the reduction of the number of infectious episodes, lesser severity of signs and symptoms and, consequently, a reduction in use of antibiotics and symptomatic drugs, less working and school days lost, less mortality and morbidity. The studies considered give positive results, confirming Pidotimod’s efficacy. Furthermore, many studies show a good safety profile of the drug, without recording serious adverse events and mutagenic potential, and a very low incidence of side effects. Pidotimod is also a more safe solution in patients subjected to vaccination, if compared to lyophilized polibacterial, which can’t be administered for thirty days before vaccination.     

Characterization of Renibacterium salmoninarum with reduced susceptibility to macrolide antibiotics by a standardized antibiotic susceptibility test

Three cohorts of juvenile and subadult Chinook salmon Oncorhynchus tshawytscha received multiple treatments with macrolide antibiotics for bacterial kidney disease (BKD) during rearing in a captive broodstock program. A total of 77 mortalities among the cohorts were screened for Renibacterium salmoninarum, the etiologic agent of BKD, by agar culture from kidney, and isolates from 7 fish were suitable for growth testing in the presence of macrolide antibiotics. The minimum inhibitory concentration (MIC) of erythromycin and azithromycin was determined by a modification of the standardized broth assay using defined medium. The American Type Culture Collection (ATCC) type strain 33209 exhibited a MIC of 0.008 microg m(-1) to either erythromycin or azithromycin. Isolates from 3 fish displayed MICs identical to the MICs for the ATCC type strain 33209. In contrast, isolates from 4 fish exhibited higher MICs, ranging between 0.125 and 0.250 microg ml(-1) for erythromycin and between 0.016 and 0.031 microg ml(-1) for azithromycin. Sequence analysis of the mutational hotspots for macrolide resistance in the 23S rDNA gene and the open reading frames of ribosomal proteins L4 and L22 found identical sequences among all isolates, indicating that the phenotype was not due to mutations associated with the drug-binding site of 23S rRNA. These results are the first report of R. salmoninarum with reduced susceptibility to macrolide antibiotics isolated from fish receiving multiple antibiotic treatments.     

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.     

Characterization of Two Macrolide Resistance-Related Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates

In analyzing the drug resistance phenotype and mechanism of resistance to macrolide antibiotics of clinical Pseudomonas aeruginosa isolates, the agar dilution method was used to determine the minimum inhibitory concentrations (MICs), and PCR (polymerase chain reaction) was applied to screen for macrolide antibiotics resistance genes. The macrolide antibiotics resistance genes were cloned, and their functions were identified. Of the 13 antibiotics tested, P. aeruginosa strains showed high resistance rates (ranging from 69.5–82.1%), and MIC levels (MIC90 > 256 μg/ml) to macrolide antibiotics. Of the 131 known macrolide resistance genes, only two genes, mphE and msrE, were identified in 262 clinical P. aeruginosa isolates. Four strains (1.53%, 4/262) carried both the msrE and mphE genes, and an additional three strains (1.15%, 3/262) harbored the mphE gene alone. The cloned msrE and mphE genes conferred higher resistance levels to three second-generation macrolides compared to two first-generation ones. Analysis of MsrE and MphE protein polymorphisms revealed that they are highly conserved, with only 1–3 amino acids differences between the proteins of the same type. It can be concluded that even though the strains showed high resistance levels to macrolides, known macrolide resistance genes are seldom present in clinical P. aeruginosa strains, demonstrating that a mechanism other than this warranted by the mphE and msrE genes may play a more critical role in the bacteria’s resistance to macrolides.Key words: Pseudomonas aeruginosa, macrolide, resistance gene, mphE, msrE     

Antibiotic resistance is prevalent in an isolated cave microbiome

Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.     

The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23S rRNA positions 2058 and 2059

Macrolides are a diverse group of antibiotics that inhibit bacterial growth by binding within the peptide tunnel of the 50S ribosomal subunit. There is good agreement about the architecture of the macrolide site from different crystallography studies of bacterial and archaeal 50S subunits. These structures show plainly that 23S rRNA nucleotides A2058 and A2059 are located accessibly on the surface of the tunnel wall where they act as key contact sites for macrolide binding. However, the molecular details of how macrolides fit into this site remain a matter of contention. Here, we have generated an isogenic set of single and dual substitutions at A2058 and A2059 in Mycobacterium smegmatis to investigate the effects of the rRNA mutations on macrolide binding. Resistances conferred to a comprehensive array of 11 macrolide compounds are used to assess models of macrolide binding predicted from the crystal structures. The data indicate that all macrolides and their derivatives bind at the same site in the tunnel with their C5 amino sugar in a similar orientation. Our data are compatible with the lactone rings of 14-membered and 16-membered macrolides adopting different conformations, enabling the latter compounds to avoid a steric clash with 2058G. This difference, together with interactions conveyed via substituents that are specific to certain ketolide and macrolide sub-classes, influences the binding to the large ribosomal subunit. Our genetic data show no support for a derivatized-macrolide binding site that has been proposed to be located further down the tunnel.     

Drug Resistance of Staphylococci

Examination of cross resistance to macrolide antibiotics in erythromycin resistant staphylococcal strains isolated from clinical sources in the United States showed that there were two types of cross resistance, group A (13.4%) and group C (86.6%). Group A possessed multiple resistance to the macrolide antibiotics, erythromycin, oleandomycin, leucomycin and spiramycin, and was also resistant to lincomycin. In group C, resistance to erythromycin alone or to both erythromycin and oleandomycin could be induced by exposure to erythromycin.     

Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli

Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.     

Peptide-mediated macrolide resistance reveals possible specific interactions in the nascent peptide exit tunnel

Expression of specific short peptides can render cells resistant to macrolide antibiotics. Peptides conferring resistance to structurally different macrolides including oleandomycin, azithromycin, azaerythromycin, josamycin and a ketolide cethromycin were selected from a random pentapeptide expression library. Analysis of the entire collection of the resistance peptides allowed their classification into five distinct groups according to their sequence similarity and the type of resistance they confer. A strong correlation was observed between the structures of macrolide antibiotics and sequences of the peptides conferring resistance. Such a correlation indicates that sequence-specific interactions between the nascent peptide and the macrolide antibiotic and/or the ribosome can occur in the ribosomal exit tunnel.