No abstract

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.Macrolide antibiotics inhibit protein synthesis in a wide range of pathogenic bacteria. The drugs bind to a single site in the large ribosomal subunit located near the entrance to the nascent peptide tunnel and are thought to sterically hinder the growth of the polypeptide chain and cause dissociation of peptidyl-tRNA from the ribosome (1, 10, 18, 33). Macrolide antibiotics were also shown to affect ribosome assembly (9).The macrolide binding site is composed primarily of RNA. Two segments of 23S rRNA, the central loop of domain V and the loop of hairpin 35 from domain II, are believed to be the major components of the drug binding site on the ribosome. Mutations of A2058 (Escherichia coli numeration) and several neighboring positions in the central loop of domain V confer resistance to macrolides (27), as reviewed in references 34 and 35. Methylation of A2058 by Erm-type methyl-transferases drastically reduces the affinity of macrolides for the ribosome, suggesting the direct interaction between this RNA residue and the drug (11,1...

The Erm family of methyltransferases is responsible for the development of resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics. These enzymes methylate an adenine of 23S ribosomal RNA that prevents the MLS antibiotics from binding to the ribosome and exhibiting their antibacterial activity. Here we describe the three-dimensional structure of an Erm family member, ErmAM, as determined by NMR spectroscopy. The catalytic domain of ErmAM is structurally similar to that found in other methyltransferases and consists of a seven-stranded beta-sheet flanked by alpha-helices and a small two-stranded beta-sheet. In contrast to the catalytic domain, the substrate binding domain is different from other methyltransferases and adopts a novel fold that consists of four alpha-helices.

Background Transmission of HIV drug resistance (TDR) gives rise to reduced efficacy of initial antiretroviral treatment, and has become a public health concern. Methods A nationwide survey on TDR was conducted in antiretroviral therapy naïve HIV-1 infected individuals from September 2004 to October 2005 in China. Drug resistance genotyping was performed on subjects’ plasma samples. Drug resistance mutations were determined and scored by Stanford HIV Drug Resistance algorithm. Results Sequences were obtained from 676 individuals, of which 61.2% were former plasma and blood donors, 17.3% were infected sexually, and 17.2% were intravenous drug users. Subtype B’ HIV-1 strains were found in 73.5%, CRF01_AE in 13.9%, CRF07_BC in 6.2%, CRF08_BC in 2.7%, subtype C in 1.04%, subtype B in 0.9%, CRF02_AG in 0.4% and B’/C intersubtype recombinant strains in 1.3% of the subjects. Twenty-six (3.8%) were found to harbor drug resistance strains. The rates of resistance to PIs, NRTIs and NNRTIs were 0.4%, 1.6% and 2.1%, respectively. Though there was no significant difference in TDR rates between 2004 and 2005 (2.9% vs. 4.4%), an increased trend was observed in the rate of high-level drug resistance (0.8% in 2004 vs. 3.0% in 2005, P = 0.0634). Conclusions The rate of TDR was relatively low in China, as compared with those in developed countries. Surveys among recently HIV-infected subjects should be preformed continually to ensure the success of the scale-up antiretroviral treatment.

Background: The coronavirus disease 19 (COVID-19) pandemic has become a global threat. Few studies have explored the risk factors for the recovery time of patients with COVID-19. This study aimed to explore risk factors associated with long-term hospitalization in patients with COVID-19.Methods: In this retrospective study, patients with laboratory-confirmed COVID-19 hospitalized in a hospital in Wuhan by March 30, 2020, were included. Demographic, clinical, laboratory, and radiological data from COVID-19 patients on hospital admission were extracted and were compared between the two groups, defined as short-and long-term hospitalization, respectively according to the median hospitalization time. Univariable and multivariable logistic regression methods were performed to identify risk factors associated with long-term hospitalization in patients with COVID-19.Results: A total of 125 discharged patients with COVID-19 were reviewed, including 123 general patients and two severe patients. The median hospitalization time was 13.0 days (IQR 10.0-17.0). Among them, 66 patients were discharged <14 days (short-term group) and 59 patients were discharged ≥14 days (long-term group). Compared with the short-term group, patients in the long-term group had significantly higher levels of C-reactive protein (P = 0.000), troponin I (P = 0.002), myoglobin (P = 0.037), aspartate aminotransferase (P = 0.005), lactic dehydrogenase (P = 0.000), prothrombin time (P = 0.030), fibrinogen (P = 0.000), and D-dimer (P = 0.006), but had significantly lower levels of lymphocyte count (P = 0.001), platelet count (P = 0.017), albumin (P = 0.001), and calcium (P = 0.000). Additionally, the incidences of hypocalcemia (P = 0.001), hyponatremia (P = 0.021), hypochloremia (P = 0.019), and bilateral pneumonia (P = 0.000) in the long-term group were significantly higher than those in the short-term group. Multivariable regression showed that hypocalcemia (P = 0.007, OR 3.313, 95% CI 1.392-7.886), hypochloremia (P = 0.029, OR 2.663, 95% CI 1.104-6.621), and bilateral pneumonia (P = 0.009, OR 5.907, 95% CI 1.073-32.521) were independent risk factors associated with long-term hospitalization in patients with COVID-19. Furthermore, a ROC curve where the area under the ROC was 0.766 for retained variables is presented.Wu et al. Risk Factors for Recovery Time of COVID-19Conclusions: Hypocalcemia, hypochloremia, and bilateral pneumonia on hospital admission were independent risk factors associated with long-term hospitalization in patients with COVID-19. To the best of our knowledge, this is the first study to highlight the importance of electrolyte imbalance in predicting the hospitalization time of patients with COVID-19.

We found a strong correlation between travel volumes departing from Wuhan, Hubei Province before the Spring Festival and the extent of amplification of the outbreak of COVID-19 in China in 2020, with 100 top cities. Almost 70% of exportations were within cities in Hubei province.

The Erm family of methyltransferases confers resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics through the methylation of 23S ribosomal RNA. Upon the methylation of RNA, the MLS antibiotics lose their ability to bind to the ribosome and exhibit their antibiotic activity. Using an NMR-based screen, we identified a series of triazine-containing compounds that bind weakly to ErmAM. These initial lead compounds were optimized by the parallel synthesis of a large number of analogues, resulting in compounds which inhibit the Erm-mediated methylation of rRNA in the low micromolar range. NMR and X-ray structures of enzyme/inhibitor complexes reveal that the inhibitors bind to the S-adenosylmethionine binding site on the Erm protein. These compounds represent novel methyltransferase inhibitors that serve as new leads for the reversal of Erm-mediated MLS antibiotic resistance.

Macrolide resistance in Streptococcus pneumoniae has been associated with two main mechanisms: target modification by Erm methyltransferases and efflux by macrolide pumps. The ketolide ABT-773, which has a 3-keto group and no L-cladinose sugar, represents a new class of drugs with in vitro activity against a variety of resistant bacteria. Several approaches were undertaken to understand how ABT-773 was able to defeat resistance mechanisms. We demonstrated tighter ribosome binding of ABT-773 than erythromycin. We also showed that ABT-773 (i) accumulated in macrolide-sensitive S. pneumoniae at a higher rate than erythromycin, (ii) was able to bind with methylated ribosomes, though at lower affinities than with wild-type ribosomes, and (iii) accumulated in S. pneumoniae strains with the efflux-resistant phenotype.Macrolide resistance in Streptococcus pneumoniae has been associated with two main mechanisms. The first resistant strains described were of the macrolide-lincosamide-streptogramin B (MLS) phenotype (18). These strains have a posttranslational modification of 23S rRNA due to the methylation of A2058 (Escherichia coli numbering system) by Erm methyltransferase, resulting in a conformational change in the ribosomes. Resistance to macrolides may be constitutively expressed or induced by sub-MICs of the macrolide (18,20). The second mechanism of resistance recently found in S. pneumoniae, and referred to as the M phenotype, results from the active efflux of 14-and 15-membered macrolides (16). The macrolide efflux pump protein in S. pneumoniae is encoded by the gene mefE (17). The prevalence of M phenotype resistance in clinical isolates of S. pneumoniae has been reported to be a significant portion (60 to 75%) of the resistance population in the United States (8, 14,15,16).The ketolide ABT-773, which has a 3-keto group and no L-cladinose sugar, represents a new class of drugs with in vitro activity against a variety of resistant phenotypes (1). The purpose of this study was to understand how ABT-773 is able to defeat these resistance mechanisms. Several approaches were undertaken, including the measurement of drug-ribosome binding kinetics, drug uptake and efflux rates, and drug effect on cell-free protein translation. MATERIALS AND METHODSChemicals and radioisotopes. Todd-Hewitt broth and yeast extract were obtained from Difco Laboratories (Detroit, Mich.) Miller's M9 medium was prepared with M9 salts purchased from Life Technologies LTD (Paisley, Scotland) but without vitamins or amino acids, as described elsewhere (10). Luciferin reagents (GenLux kit) were purchased from Wallac (Turku, Finland). All other chemicals, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), were purchased from Sigma Chemical Co. (St. Louis, Mo.). [N-methyl-14 C]erythromycin (55 mCi/mmol) was obtained from NEN Life Science Products (Boston, Mass.), and unlabeled erythromycin was made in-house. [N-methyl-14 C]ABT-773 (27 mCi/mmol) was prepared in-house by coupling 6-0-allyl ketolide and 3-[U-14 C]bromoquinoline according to a...