Structure–Activity Relationships for Six Ketolide Antibiotics

Six structurally related 3-keto-substituted macrolide antibiotics (ketolides) were compared for concentration-dependent inhibitory effects on growth rate, viable cell number, and protein synthesis rates in Staphylococcus aureus cells. Inhibitory effects on 50S ribosomal subunit formation were also examined, as this is a second target for these antibiotics. A concentration range of 0.01 to 0.1 microg/ml was tested. An IC50 for inhibition of translation and 50S synthesis was measured for each compound, to relate structural features to inhibitory activity. ABT-773 was the most effective of the six compounds tested with an IC50 = 0.035 microg/ml. HMR 3004 was almost as effective with an IC50 = 0.05 microg/ml. Two 2-fluoroketolides (HMR 3562 and HMR 3787) were equivalent in their inhibitory activity with an IC50 = 0.06 microg/ml. Telithromycin (HMR 3647) had an IC50 = 0.08 microg/ml, and HMR 3832 was least effective with an IC50 = 0.11 microg/ml. Each antibiotic had an equivalent inhibitory effect on translation and 50S subunit formation. These results indicate specific structural features of these antimicrobial agents, which contribute to defined inhibitory activities against susceptible organisms.     

Conformations in solution and bound to bacterial ribosomes of ketolides, HMR 3647 (telithromycin) and RU 72366: a new class of highly potent antibacterials

The new class of antibiotics called ketolides is endowed with remarkable antibacterial activity against macrolide-resistant strains. Further modifications of the 3 keto-macrolactone backbone led to 11,12-hydrazonocarbamate ketolides with an imidazolyl pyridine chain: the file-leader of ketolide class, HMR 3647 (telithromycin), and its N-bis-demethyl-derivative, RU 72366. The potency of HMR 3647 is higher than that of RU 72366. Stereospecific 1H and 13C resonance assignments of HMR 3647 and RU 72366 have been determined and have allowed a detailed quantitative conformational analysis of the uncomplexed form of the molecules. The comparative conformation of HMR 3647 in solution and its N-bis-demethyl-derivative in D2O has been carried out using different heteronuclear correlation experiments in conjunction with nuclear Overhauser effect experiments and in particular long-range 3J(CH) coupling constants and using molecular dynamics (MD) methods. The study of ketolide ribosome interaction has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The database of ribosome-bound ketolide structures has been used to compare the structure(s) of ketolide in ribosome-ketolide complexes with the conformational preferences of free ketolides and to highlight the significant differences between HMR 3647 and RU 72366. A comparison of the conformations bound to ribosome was made with those of other previously studied ketolide (RU 004) and macrolides and would explain the remarkable potencies of HMR 3647 in inhibiting protein synthesis.     

Upper respiratory tract microbial communities, acute otitis media pathogens, and antibiotic use in healthy and sick children

The composition of the upper respiratory tract microbial community may influence the risk for colonization by the acute otitis media (AOM) pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. We used culture-independent methods to describe upper respiratory tract microbial communities in healthy children and children with upper respiratory tract infection with and without concurrent AOM. Nasal swabs and data were collected in a cross-sectional study of 240 children between 6 months and 3 years of age. Swabs were cultured for S. pneumoniae, and real-time PCR was used to identify S. pneumoniae, H. influenzae, and M. catarrhalis. The V1-V2 16S rRNA gene regions were sequenced using 454 pyrosequencing. Microbial communities were described using a taxon-based approach. Colonization by S. pneumoniae, H. influenzae, and M. catarrhalis was associated with lower levels of diversity in upper respiratory tract flora. We identified commensal taxa that were negatively associated with colonization by each AOM bacterial pathogen and with AOM. The balance of these relationships differed according to the colonizing AOM pathogen and history of antibiotic use. Children with antibiotic use in the past 6 months and a greater abundance of taxa, including Lactococcus and Propionibacterium, were less likely to have AOM than healthy children (odds ratio [OR], 0.46; 95% confidence interval [CI], 0.25 to 0.85). Children with no antibiotic use in the past 6 months, a low abundance of Streptococcus and Haemophilus, and a high abundance of Corynebacterium and Dolosigranulum were less likely to have AOM (OR, 0.51; 95% CI, 0.31 to 0.83). An increased understanding of polymicrobial interactions will facilitate the development of effective AOM prevention strategies.     

Microbiological evaluation of antibiotics for empirical therapy of community-acquired infections of the lower respiratory tract]

During first 3 days after patient hospitalization with pneumonia or chronic obstruction pulmonary disease (COPD) pathogens in sputum were studied according NCCLS standards (for 1999 year). Among 93 pathogens isolated in pneumonia the most frequent were S. pneumoniae (41.9%), H. influenzae (21.5%). Among 232 pathogens isolated in COPD the most frequent were S. pneumoniae (35.5%), H. influenzae (16.8%). Other pathogens were staphylococci, moraxella, gram-negative bacteria. No penicillin-resistant S. pneumoniae, were isolated, the strains with moderate penicillin resistance were less than 3% in both groups. Among H. influenzae isolated from patients with pneumonia 25% were beta-lactamase producers, from COPD patients 21% strains produced beta-lactamase. Totally among all studied pathogens only 58% were sensitive to ampicillin in pneumonia groups and 48% in COPD groups, for azithromycin 70.7% and 71% respectively, for cefuroxime 84.5% and 85% respectively. Ampicillin efficacy for empirical treatment of community-acquired low respiratory tract infections was substantially less than that of modern antibiotics.     

A new type of ketolide bearing an N-aryl-alkyl acetamide moiety at the C-9 iminoether: synthesis and structure-activity relationships

A new type of ketolide bearing an N-aryl-alkyl acetamide moiety at the C-9 iminoether and its analogues were prepared, and their antibacterial activities and pharmacokinetic properties were evaluated. We found that the introduction of an (R)-alkyl group between the amide and iminoether groups could improve the pharmacokinetic properties while maintaining the activity against erythromycin-resistant Streptococcus pneumoniae. Among the ketolides prepared with the (R)-alkyl group, compound 5p with an N-(3-quinoxalin-6-yl-propyl)-propionamide moiety was found to have in vivo efficacy comparable to CAM with potent in vitro antibacterial activities against the key respiratory pathogens including Haemophilus influenzae and erythromycin-resistant S. pneumoniae.     

Role of p38 MAP kinase and transforming growth factor-beta signaling in transepithelial migration of invasive bacterial pathogens

Streptococcus pneumoniae and Haemophilus influenzae are human pathogens that often asymptomatically colonize the mucosal surface of the upper respiratory tract, but also occasionally cause invasive disease. The ability of these species to traverse the epithelium of the airway mucosa was modeled in vitro using polarized respiratory epithelial cells in culture. Migration across the epithelial barrier was preceded by loss of transepithelial resistance. Membrane products of S. pneumoniae that included lipoteichoic acid induced disruption of the epithelial barrier in a Toll-like receptor 2-dependent manner. This result correlates with a recent genetic study that associates increased TLR2 signaling with increased rates of invasive pneumococcal disease in humans. Loss of transepithelial resistance by the TLR2 ligand correlated with activation of p38 MAP kinase and transforming growth factor (TGF)-beta signaling. Activation of p38 MAPK and TGF-beta signaling in epithelial cells upon nasal infection with S. pneumoniae was also demonstrated in vivo. Inhibition of either p38 MAPK or TGF-beta signaling was sufficient to inhibit the migration of S. pneumoniae or H. influenzae. Our data shows that diverse bacteria utilize common mechanisms, including MAPK and TGF-beta signaling pathways to disrupt epithelial barriers and promote invasion.     

Synthesis and antibacterial activity of C12 des-methyl ketolides

Synthesis of C(12) des-methyl ketolide is developed featuring an intramolecular epoxide formation/elimination process to establish the C(12) stereocenter. These ketolides are potent against several key respiratory pathogens, including erythromycin resistant erm- and mef-containing strains of Streptococcus pneumoniae.     

Discovery of a compound which acts as a bacterial UMP kinase PyrH inhibitor

PyrH is a member of the UMP kinase family that catalyses the conversion of UMP to UDP, an essential step in the pyrimidine metabolic pathway in a variety of bacteria including those causing community-acquired respiratory tract infections (RTIs). In this study, we have developed a luminescence-based kinase assay of PyrH and evaluated the inhibitory activity of PYRH-1 (sodium {3-[4-tert-butyl-3-(9H-xanthen-9-ylacetylamino)phenyl]-1-cyclohexylmethylpropoxycarbonyloxy}acetate). PYRH-1 inhibits PyrH derived from both Streptococcus pneumoniae and Haemophilus influenzae with IC(50) (concentration of inhibitor giving a 50% decrease in enzyme activity) values of 48 and 75?μM, respectively, whose inhibitory activity against S.?pneumoniae PyrH was far higher compared with that of UTP (IC(50) =?710?μM), an allosteric PyrH inhibitor. The molecular interaction analysis by surface plasmon resonance suggested that PYRH-1 directly interacts with S.?pneumoniae PyrH at one-to-one molar ratio. Finally, PYRH-1 was shown to have antimicrobial activity against several different bacteria causing RTIs, such as S.?pneumoniae, Staphylococcus aureus, H.?influenzae (acrA knockout strain), suggesting that PYRH-1 is a prototype chemical compound that can be harnessed as an antimicrobial drug with a novel mode of action by targeting bacterial PyrH.     

Induction of murine B cell proliferation and immunoglobulin synthesis by some bacterial ribosomes

Ribosomal preparations from Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Streptococcus pneumoniae were investigated with respect to their activating capacity towards murine lymphoid cells. The proliferation of BALB/c spleen cells was induced in a dose-dependent fashion (from 1 to 100 micrograms/ml) by ribosomes of K. pneumoniae, H. influenzae, and S. pyogenes with a peak activity at 48 or 72 hr of culture. The majority of the blast cells induced by these ribosomal preparations were positive for surface-immunoglobulin (S-Ig) and negative for Thy 1.2. Furthermore, K. pneumoniae, H. influenzae, and S. pyogenes ribosomes induced the synthesis of IgM and some IgA. Cell proliferation and induction of IgM production were also demonstrated with the 3 ribosomal preparations using spleen cells from athymic nude (nu+/nu+) mice, Lyb-5-defective CBA/N spleen cells, B cell-enriched and T cell-depleted BALB/c spleen cell suspensions, as well as spleen cells from the Ips gene-deficient C3H/HeJ strain. Cell culture supernatants contained specific anti-ribosome IgM antibodies. Antibodies of other specificities (anti-sheep erythrocytes) were also demonstrated in supernatants from K. pneumoniae-stimulated cultures. Evidence against a possible role of contamination of K. pneumoniae and H. influenzae ribosomes by lipopolysaccharide- or lipid A-associated proteins in this effect is discussed. Ribosomes from S. pneumoniae did not induce 3H-thymidine incorporation nor Ig production. None of the 4 ribosomal preparations was found to stimulate T cell blastogenesis or to induce interleukin-2 production by naive BALB/c spleen cells. Finally, ribosomes from H. influenzae, S. pyogenes, S. pneumoniae but not those of K. pneumoniae stimulated interleukin-1 production by adherent spleen cells, from BALB/c mice.     

New antibacterial tetrahydro-4(2H)-thiopyran and thiomorpholine S-oxide and S,S-dioxide phenyloxazolidinones

Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.     

Stimulation of bacterial adherence by neutrophil defensins varies among bacterial species but not among host cell types

Adherence of Haemophilus influenzae to bronchial epithelial cells is enhanced by neutrophil defensins, which are released from activated neutrophils during inflammation [Gorter et al. (1998) J. Infect. Dis. 178, 1067-1078]. In this study, we showed that the adherence of H. influenzae to various epithelial, fibroblast-like and endothelial cell types was significantly enhanced by defensins (20 microg ml(-1)). Defensins stimulated also the adherence of Moraxella catarrhalis, Neisseria meningitidis and nonencapsulated Streptococcus pneumoniae to the NCI-H292 cell line. In contrast, defensins did not affect the adherence of Pseudomonas aeruginosa, encapsulated S. pneumoniae, Escherichia coli and Staphylococcus epidermidis. These results suggest that the defensin-enhanced adherence might support the adherence and possibly persistence of the selected bacterial species using the respiratory tract as port of entry.     

Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria

During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defence. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signalling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabelled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus alpha-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.     

Rational use of antibiotics in mucoviscidosis pediatric cases with an account of local microbiological monitoring]

The results of local microbiological monitoring of bronchial secretion in 482 children with mucoviscidosis observed within 2000-2004 in the Republican Centre of Mucoviscidosis are presented. The results provided development of recommendations for rational use of antibiotics in the treatment of infectious processes in pediatric patients with mucoviscidosis. Since the emergence of MRSA in such patients is low, it is recommended to use antistaphylococcal betalactams (oxacillin, cefazolin, amoxycillin/clavulanate) for the treatment of infections due to S. aureus. For the treatment of infections due to some other pathogens, except S. maltophilia, the most active betalactams were carbapenems (imipenem and meropenem). Ciprofloxacin was active against numerous etiological agents causing low respiratory tract infections in children with mucoviscidosis except S. maltophila and A. xylosoxidans subsp. xylosoxidans. For the treatment of infections due to P. aeruginosa, P. aeruginosa muc. and K. pneumoniae the most active aminoglycosides were amikacin and tobramycin (for P. aeruginosa and P. aeruginosa muc.), while gentamicin was not active in such cases. As for antibiotics of other groups, high activity against S. aureus in the treatment of children with mucoviscidosis was recarded with the use of vancomycin, fusidic acid and rifampicin. Azithromycin and co-trimoxazole were active against H. influenzae. Chloramphenicol was active against S. maltophilia, B. cepacia and H. influenzae in the treatment of such patients.     

Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges

The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.     

Bacterial infectious process in patients with chronic obliterating bronchiolitis

Chronic obliterating bronchiolitis in children is characterized by the presence of chronic multifactor inflammatory infectious process with Streptococcus pneumoniae and Haemophilus influenzae being the leading causative agents. The inflammatory process is induced and maintained by S. pneumoniae and H. influenzae or by the association of these two pathogens, but the severity of the clinical course of obliterating bronchiolitis is determined by H. influenzae, which is confirmed by its high detection rate at the phase of exacerbation in patients with pronounced bronchial obstruction.     

上呼吸道感染患儿咽部病原菌分布及耐药性分析

目的了解天津滨海地区儿童上呼吸道感染病原菌情况及耐药性,以便指导临床合理用药。方法对2012年1月至2013年4月天津市滨海新区汉沽中医医院住院儿科1022例上呼吸道感染儿童咽拭子标本进行培养及鉴定。采用法国ATB自动细菌鉴定仪及MIC药敏板进行细菌鉴定和药敏试验,β-内酰胺酶试验采用头孢硝噻吩纸片法,耐甲氧西林金黄色葡萄球菌（MRSA）采用头孢西丁纸片法检测。结果检出病原菌296株,其中金黄色葡萄球菌、肺炎链球菌及流感嗜血杆菌检出率分别为13. 70%、5. 97% 、5. 19%,分列前三位。金黄色葡萄球菌对常用抗菌药物耐药率为苯唑西林30. 0%、青霉素95. 7%、红霉素80. 0%、左氧氟沙星4. 3%,MRSA占30. 0% （42/140）、;肺炎链球菌对常用抗菌药物耐药率为青霉素9. 84%、红霉素54. 1%、克林霉素52.5%、左氧氟沙星3. 3% ;流感嗜血杆菌对常用抗菌药物耐药率为氨苄西林34. 0%、阿奇霉素0%、左氧氟沙星0%,复方新诺明56. 6%、头孢呋辛3. 8%、阿莫西林/克拉维酸5. 7%。结论天津滨海地区儿童上呼吸道感染病原菌分布广泛,检出致病菌有一定的耐药率,临床应重视根据病原菌感染情况及药敏试验结果,进行耐药性监测和合理用药。     

Synthesis and antibacterial activity of 6-O-(heteroaryl-isoxazolyl)propynyl 2-fluoro ketolides

Macrolide antibiotics are widely prescribed for the treatment of respiratory tract infections; however, the increasing prevalence of macrolide-resistant pathogens is a public health concern. Therefore, the development of new macrolide derivatives with activities against resistant pathogens is urgently needed. A series of novel 6-O-(heteroaryl-isoxazolyl)propynyl 2-fluoro ketolides has been synthesized from erythromycin A. These compounds have shown very promising in vitro and in vivo antibacterial activities against key respiratory pathogens including erythromycin-susceptible/resistant strains.     

In-vitro Comparison of Erythromycin,Lincomycin, and Clindamycin

The in-vitro antibacterial activities of erythromycin, lincomycin, and clindamycin, a new derivative of lincomycin, were compared. Clindamycin was always more active than lincomycin, and was either as active as erythromycin or more so against betahaemolytic streptococci, Streptococcus viridans, Str. pneumoniae, and erythromycin-sensitive Staphylococcus aureus. It was also fully active against most erythromycin-resistant strains of Staph. aureus. On the other hand, it was somewhat less active than erythromycin against Haemophilus influenzae and considerably less active than erythromycin against Str. faecalis and Neisseria gonorrhoeae.Clinical trials seem to be justified in infections with sensitive organisms for which erythromycin might have been indicated.     

Serum bactericidal activities of moxifloxacin and levofloxacin against aerobic and anaerobic intra-abdominal pathogens

We studied the serum bactericidal activity (SBA) of moxifloxacin and levofloxacin against common pathogens associated with complicated intra-abdominal infections. Ten healthy volunteers received a single dose of moxifloxacin (400 mg) and levofloxacin (750 mg) and serum samples were collected at 2, 4, 8, 12, and 24h after the dose of each drug. Bactericidal titers in serum over time were determined for aerobic gram-negative bacilli (Escherichia coli, Klebseilla pneumoniae, and Enterobacter cloacae) and anaerobic bacteria (Bacteroides fragilis, Bacteroides thetaiotaomicron, Prevotella bivia, and Finegoldia magna). Both fluoroquinolones provided rapid (2h) attainment and prolonged (24h) SBA (titers > or = 1:8) against each of the aerobic bacilli studied. SBA was observed for at least 12h against B. fragilis strains with MICs < or = 2 microg/ml to moxifloxacin and < or = 4 microg/ml to levofloxacin. Prolonged (12h) SBA (titers > or = 1:2) was also observed against isolates of B. thetaiotaomicron, P. bivia, and F. magna with moxifloxacin < or = MICs 2 microg/ml.     

Signature patterns revealed by microarray analyses of mice infected with influenza virus A and Streptococcus pneumoniae

We used cDNA microarrays to identify differentially expressed genes in mice in response to infections with influenza virus A/PR/8/34 (H1N1) and Streptococcus pneumoniae. Expression microarray analysis showed up-regulation and down-regulation of many genes involved in the defense, inflammatory response and intracellular signaling pathways including chemokine, apoptosis, MAPK, Notch, Jak-STAT, T-cell receptor and complement and coagulation cascades. We have revealed signature patterns of gene expression in mice infected with two different classes of pathogens: influenza virus A and S. pneumoniae. Quantitative real-time RT-PCR results confirmed microarray results for most of the genes tested. These studies document clear differences in gene expression profiles between mice infected with influenza virus A and S. pneumoniae. Identification of genes that are differentially expressed after respiratory infections can provide insights into the mechanisms by which the host interacts with different pathogens, useful information about stage of diseases and selection of suitable targets for early diagnosis and treatments. The advantage of this novel approach is that the detection of pathogens is based on the differences in host gene expression profiles in response to different pathogens instead of detecting pathogens directly.