Search

Objective. To reveal the prevalence of antimicrobial resistance of H. influenzae, S. pneumoniae, and M. catarrhalis clinical pediatric isolates in Yekaterinburg and Sverdlovsk region during 2013-2015. Materials and Methods. In total 231 H. influenzae, 289 S. pneumoniae, and 266 M. catarrhalis isolates were included in the study. Antimicrobial susceptibility testing was performed partially by disc-diffusion method and partially by automated method, depending on the local practice; β-lacamase production was detected by the nitrocefin disc test. Results. Among H. influenzae isolates 211 (91.4%) were β-lactamase-negative and susceptible to ampicillin, 13 (5.6%) β-lactamase-positive and resistant to ampicillin; 7 (3%) – β-lactamase-negative and resistant to ampicillin. Among S. pneumoniae strains 33.2% were non-susceptible to penicillin; susceptibility to cefotaxime and ceftriaxone was 89.2% and 93.5% respectively; 27.3% of strains were resistant to erythromycin, 20.8% – to clindamycin. β-Lactamase production was detected in 91.7% of M. catarrhalis isolates. Conclusions. H. influenzae remain very high susceptibility level to β-lactams. Increase of the prevalence of S. pneumoniae non-susceptibility to penicillin and other β-lactams require further monitoring. High rate of β-lactamase production by M. catarrhalis isolates was noted.

The mRNA coding sequence defines not only the amino acid sequence of the protein, but also the speed at which the ribosomes move along the mRNA while making the protein. The non-uniform local kinetics - denoted as translational rhythm - is similar among mRNAs coding for related protein folds. Deviations from this conserved rhythm can result in protein misfolding. In this review we summarize the experimental evidence demonstrating how local translation rates affect cotranslational protein folding, with the focus on the synonymous codons and patches of charged residues in the nascent peptide as best-studied examples. Alterations in nascent protein conformations due to disturbed translational rhythm can persist off the ribosome, as demonstrated by the effects of synonymous codon variants of several disease-related proteins. Charged amino acid patches in nascent chains also modulate translation and cotranslational protein folding, and can abrogate translation when placed at the N-terminus of the nascent peptide. During cotranslational folding, incomplete nascent chains navigate through a unique conformational landscape in which earlier intermediate states become inaccessible as the nascent peptide grows. Precisely tuned local translation rates, as well as interactions with the ribosome, guide the folding pathway towards the native structure, whereas deviations from the natural translation rhythm may favor pathways leading to trapped misfolded states. Deciphering the 'folding code' of the mRNA will contribute to understanding the diseases caused by protein misfolding and to rational protein design., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The ribosome can slide along mRNA without establishing codon-anticodon interactions. This movement can be regulated (programmed) by the elements encoded in the mRNA, as observed in bypassing of non-coding gap in gene 60 of bacteriophage T4, or occur spontaneously, such as during traversal by the 70S ribosome of the 3'UTRs or upon re-initiation on bacterial polycistronic genes. In this study, we investigate the kinetic mechanism underlying the programmed and spontaneous ribosome sliding. We show that the translation rate of gene 60 mRNA decreases as the ribosome approaches the take-off site, especially when the KKYK regulatory sequence in the nascent peptide reaches the constriction site in the ribosome exit tunnel. However, efficiency of bypassing increases when the ribosome traverses the gap quickly. With the non-coding gap exceeding the natural 50 nt, the processivity of sliding remains high up to 56 nt, but drops sharply beyond that due to the loss of mRNA elements support. Sliding efficiency is temperature-dependent; while temperature regulates the number of ribosomes initiating programmed bypassing, traversing the long gaps becomes increasingly unfavorable at lower temperatures. This data offers novel insights into the kinetic determinants of programmed and spontaneous ribosome sliding along the mRNA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the exit port where it emerges into the cytoplasm. The ribosome defines the unique energy landscape of the pioneering round of protein folding. The spatial confinement and the interactions of the nascent peptide with the tunnel walls facilitate formation of secondary structures, such as α-helices. The vectorial nature of protein folding inside the tunnel favors local intra- and inter-molecular interactions, thereby inducing cotranslational folding intermediates that do not form upon protein refolding in solution. Tertiary structures start to fold in the lower part of the tunnel, where interactions with the ribosome destabilize native protein folds. The present review summarizes the recent progress in understanding the driving forces of nascent protein folding inside the tunnel and at the surface of the ribosome., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a β-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a β-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row. The downstream errors in a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a distinct class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs.

The quality of culture media for blood culture was checked: nutrient medium for children with an antibiotic neutralizer for the cultivation of aerobes, nutrient medium with an antibiotic neutralizer for the cultivation of anaerobes, a nutrient medium with an antibiotic neutralizer for the cultivation of aerobes, nutrient medium for the cultivation of aerobes UNONA® used in the automatic bacteriological analyzer JUNONA ®Labstar 50 (SCENKER Biological Technology Co., Ltd. China). Used tenfold dilutions from 18-24 hour cultures of reference strains: ATCC 13124 Clostridium perfringens; ATCC 25285 Bacteroides fragilis; NCTC 194I8 Haemophilus influenzae; ATCC 49619 Streptococcus pneumoniae; ATCC 16615 Streptococcus pyogenes; ATCC 27853 Pseudomonas aeruginosa; ATCC 25923 Staphylococcus aureus; ATCC 25922 Escherichia coli; BKPGU-401/-885-653 Candida albicans; ATCC13813 Streptococcus agalactiae; No. 186 Enterobacter cloacae; ATCC 29212 Enterococcus faecalis; clinical isolates: Acinetobacter lwofii, Enterobacter cloacae, Candida tropicalis. All investigated reference strains were isolated on nutrient media in accordance with their biological properties when inoculated with 50 CFU / ml less than 72 hours later, as stated by the manufacturer. The study has shown that growth factors must be used to test the quality of the culture media with Haemophilus influenzae bacteria and this must be reflected in the manufacturer's instructions., Competing Interests: The authors declare no conflict of interest.

Protein homeostasis of bacterial cells is maintained by coordinated processes of protein production, folding, and degradation. Translational efficiency of a given mRNA depends on how often the ribosomes initiate synthesis of a new polypeptide and how quickly they read the coding sequence to produce a full-length protein. The pace of ribosomes along the mRNA is not uniform: periods of rapid synthesis are separated by pauses. Here, we summarize recent evidence on how ribosome pausing affects translational efficiency and protein folding. We discuss the factors that slow down translation elongation and affect the quality of the newly synthesized protein. Ribosome pausing emerges as important factor contributing to the regulatory programs that ensure the quality of the proteome and integrate the cellular and environmental cues into regulatory circuits of the cell., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Samatova, Daberger, Liutkute and Rodnina.)

Resistance of representatives of the order Enterobacterales to ertapenem 12.1%. The highest frequency of insensitivity to this antimicrobial drug was noted among isolates of K. pneumoniae 29.4%. Among all enterobacterial isolates, resistance to imipenem and meropenem was 17.2% and 20%. The proportion of P. aeruginosa strains is 50.9% resistant to meropenem and imipenem, respectively, and 45% to doripenem. In turn, A. baumannii is resistant to meropenem - 66.6%, imipenem - 63.6%, doripenem - 83.3%. The following resistance genes were found in K. pneumoniae: NDM (n=2), KPC (n=10), OXA (n=1); in P. aeruginosa: VIM (n=8), NDM (n=1), OXA (n=1); A. baumannii OXA (n=1). At present, it is optimal to use molecular methods, in particular real-time PCR, to effectively monitor the distribution of carbapenemase producers, which tend to be widely distributed in a hospital setting. Molecular methods allow you to quickly get the result (during the working day) and give an adequate decision on antibiotic therapy., Competing Interests: The authors declare no conflict of interest.

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain's hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates., Competing Interests: ML, MM, ES, JE, MR No competing interests declared, (© 2020, Liutkute et al.)

Efficient translational bypassing of a 50-nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem-loop 5' of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem-loop 5' of the take-off codon and allowing greater peptidyl-tRNA drop off. A ribosome profiling analysis of phage T4-infected Escherichia coli yielded protected mRNA fragments within the normal size range derived from ribosomes stalled at the take-off codon. However, ribosomes at this position also yielded some 53-nucleotide fragments, 16 longer. These were due to protection of the nucleotides that form the 5' stem-loop. NMR shows that the 5' stem-loop is highly dynamic. The importance of different nucleotides in the 5' stem-loop is revealed by mutagenesis studies. These data highlight the significance of the 5' stem-loop for the 50-nt bypassing and further enhance appreciation of relevance of the extent of ribosome loading for recoding., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, -1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many proteins in the cell fold cotranslationally within the restricted space of the polypeptide exit tunnel or at the surface of the ribosome. A growing body of evidence suggests that the ribosome can alter the folding trajectory in many different ways. In this review, we summarize the recent examples of how translation affects folding of single-domain, multiple-domain and oligomeric proteins. The vectorial nature of translation, the spatial constraints of the exit tunnel, and the electrostatic properties of the ribosome-nascent peptide complex define the onset of early folding events. The ribosome can facilitate protein compaction, induce the formation of intermediates that are not observed in solution, or delay the onset of folding. Examples of single-domain proteins suggest that early compaction events can define the folding pathway for some types of domain structures. Folding of multi-domain proteins proceeds in a domain-wise fashion, with each domain having its role in stabilizing or destabilizing neighboring domains. Finally, the assembly of protein complexes can also begin cotranslationally. In all these cases, the ribosome helps the nascent protein to attain a native fold and avoid the kinetic traps of misfolding.

Some proteins are expressed as a result of a ribosome frameshifting event that is facilitated by a slippery site and downstream secondary structure elements in the mRNA. This review summarizes recent progress in understanding mechanisms of -1 frameshifting in several viral genes, including IBV 1a/1b, HIV-1 gag-pol, and SFV 6K, and in Escherichia coli dnaX. The exact frameshifting route depends on the availability of aminoacyl-tRNAs: the ribosome normally slips into the -1-frame during tRNA translocation, but can also frameshift during decoding at condition when aminoacyl-tRNA is in limited supply. Different frameshifting routes and additional slippery sites allow viruses to maintain a constant production of their key proteins. The emerging idea that tRNA pools are important for frameshifting provides new direction for developing antiviral therapies., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A hallmark of translation in human immunodeficiency virus type 1 (HIV-1) is a -1 programmed ribosome frameshifting event that produces the Gag-Pol fusion polyprotein. The constant Gag to Gag-Pol ratio is essential for the virion structure and infectivity. Here we show that the frameshifting efficiency is modulated by Leu-tRNALeu that reads the UUA codon at the mRNA slippery site. This tRNALeu isoacceptor is particularly rare in human cell lines derived from T-lymphocytes, the cells that are targeted by HIV-1. When UUA decoding is delayed, the frameshifting follows an alternative route, which maintains the Gag to Gag-Pol ratio constant. A second potential slippery site downstream of the first one is normally inefficient but can also support -1-frameshifting when altered by a compensatory resistance mutation in response to current antiviral drug therapy. Together these different regimes allow the virus to maintain a constant -1-frameshifting efficiency to ensure successful virus propagation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The results of the bacteriological catheter investigation on the analyzer with the technology of laser light scattering and using D. Maki culture technique coincided in 91.8% of cases. Catheter-associated infections are proven in 8 cases. The following obtained from blood and intravascular catheter in 5 patients: Staphylococcus epidermidis (n=1), Staphylococcus haemolyticus (n=1), Escherichia coli (n=1), Staphylococcus aureus (n=2); Staphylococcus epidermidis (n=1) was detected in 1 child from ventricular shunt and cerebrospinal fluid and Escherichia coli lactose-negative + Staphylococcus haemolyticus were detected in 1 child from ventricular shunt, whily only Escherichia coli lactose-negative was detected from cerebrospinal fluid; Pseudomonas aeruginosa (n=1) was foud out in 1 patient from the urinary catheter and urine. Clinical significance of the isolated microorganisms from the catheter must be assessed in each particular case taking into account its quantity and type of the isolated microorganism., Competing Interests: The authors declare no conflict of interest.

Primarily in the study of bacteremia, coagulase-negative staphylococci and representatives of the order Enterobacterales were found. To establish the etiological role of coagulase-negative staphylococcus in making a diagnosis of sepsis in each particular case, it is necessary to consider the condition and age of the child, as well as indicators of markers of systemic inflammation. In 1/3 cases of coagulase-negative staphylococcus indicate colonization of the catheter. Staphylococcus aureus in bacteremia and sepsis - 6.5%, Haemophilus influenzae - 0.6%, Esherichia coli - 7.8%, Streptococcus agalactiae - 2%. For the diagnosis of sepsis, it is necessary to conduct repeated (at least two times) blood culture studies using high-quality nutrient media containing all the necessary growth factors, followed by a mandatory determination of the susceptibility of the isolated strains of microorganisms to antimicrobial agents. Bacteremia as a whole in children of a multidisciplinary hospital amounted to 5.8%. In premature babies, bacteremia was detected in 4.4% of cases, of which sepsis was confirmed in 41,2%., Competing Interests: The authors declare no conflict of interest.

The toxoplasmosis is a disease representing a potential risk for fetus. This is the reason to detect antibodies to agent of the given infection in case of any suspicion to intrauterine infection. The analysis was applied to 65 samples of blood serum of newborns, children of the frst year of life and pregnant women. The IgG antibodies were detected in 21.5% and 26.2% of individuals examined by test-systems Euroimmun AG and Virion-Serion. The IgM antibodies were detected in 9.4% of individuals examined by test systems of both manufacturers. To make verifcation of the results of detection of antibodies to toxoplasmosis even more reliable avidity of specifc IgG antibodies is to be analyzed., Competing Interests: The authors declare no conflict of interest.

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNA Gly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.

The gene product 60 (gp60) of bacteriophage T4 is synthesized as a single polypeptide chain from a discontinuous reading frame as a result of bypassing of a non-coding mRNA region of 50 nucleotides by the ribosome. To identify the minimum set of signals required for bypassing, we recapitulated efficient translational bypassing in an in vitro reconstituted translation system from Escherichia coli. We find that the signals, which promote efficient and accurate bypassing, are specified by the gene 60 mRNA sequence. Systematic analysis of the mRNA suggests unexpected contributions of sequences upstream and downstream of the non-coding gap region as well as of the nascent peptide. During bypassing, ribosomes glide forward on the mRNA track in a processive way. Gliding may have a role not only for gp60 synthesis, but also during regular mRNA translation for reading frame selection during initiation or tRNA translocation during elongation.

The objective of the present study was to elucidate the etiopathological significance of various Streptococcus pneumoniae serotypes in the children presenting with ENT infections and carrying nasopharyngeal pathogens. The incidence of the latter condition was 19.5% in the children free from S. pneumoniae infection in comparison with 20.9% and 30.7% in those having diagnosis of otitis media and rhinosinusitis respectively. Fifty five (88.8%) of the 62 isolated streptococcal strains were grouped into types with the use of multiplex PCR. Twelve serotypes were identified in the patients presenting with rhinosinusitis with the predominance of 6A/6B and 3 (40.5%) compared with seven isolated from the carriers of nasopharyngeal pathogens. In this group, type 3 also prevailed (26.5%) whereas other serotypes occurred less frequently: 23F (13,4%), indivisible totality of 8, 9V, 9A, 1F, 11A, 211B, 11C, 11D, 12F, 15A, and 33F (13.4%), 20 (6.7%), 19A (6.7%), 14 (6.7%), 6A,6B (6.7%). The serotypes of S. pneumoniae isolated from the patients with rhinosinusitis were found to show 55.3% identity with those present in the composition of the conjugated 7-valent pneumococcal vaccines, 63.2% identity with the 10-valent vaccine, 81.6% identity with the 11p-valnet vaccine, and 84.2% identity with the 13-valent vaccine.

Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins-carbonic anhydrase B-is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations.