Genomic surveillance as a scalable framework for precision phage therapy against antibiotic-resistant pathogens.

Phage therapy is gaining increasing interest in the fight against critically antibiotic-resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here, we combine large-scale phylogeographic analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii, a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within 6 years. As we demonstrate in Eastern Europe, this spatiotemporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of phage cocktails against prevalent strain types using in vitro and animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy.

Experimental Determination of a Chiral Ternary Solubility Diagram and Its Interpretation in Gas Antisolvent Fractionation.

Although crystallization has been widely applied for the enantiomeric enrichment of non-racemates both in research and in industrial applications, the physical-chemical background of chiral crystallizations is not as frequently discussed. A guide for the experimental determination of such phase equilibrium information is lacking. In the current paper, the experimental investigation of chiral melting phase equilibria, chiral solubility phase diagrams and their application in atmospheric and supercritical carbon dioxide-assisted enantiomeric enrichment is described and compared. Benzylammonium mandelate is a racemic compound; it shows eutectic behavior when molten. A similar eutonic composition was observed in its methanol phase diagram at 1 °C. The influence of the ternary solubility plot could be unequivocally discovered in atmospheric recrystallization experiments, which proved that the crystalline solid phase and the liquid phase were in an equilibrium. The interpretation of the results obtained at 20 MPa and 40 °C, using the methanol-carbon dioxide mixture as a pseudo-component, was more challenging. Although the eutonic composition was found to be the limiting enantiomeric excess value in this purification process as well, the high-pressure gas antisolvent fractionation results were only clearly thermodynamically controlled in certain concentration ranges.

Invasive Fungal Infection in Romania: Changing Incidence and Epidemiology During Six Years of Surveillance in a Tertiary Hospital.

The present study aimed to evaluate the incidence of candidemia, the trend of species distribution and the antifungal susceptibility patterns of all invasive strains of Candida spp. isolated over a 6-year period in an Eastern European University hospital. A total number of 156 isolates were reidentified by MALDI-TOF and tested for susceptibility by SensititreTM YeastOne™. Isolates were assigned as non-susceptible or belonging to the wild type according to the new CLSI (2017, 2018) break points and epidemiological cut-off values. C. parapsilosis (37.82%) was the most frequently isolated yeast, followed by C. albicans (26.28%). The general tendency of the species distribution during the surveyed period shifted towards an increase in C. parapsilosis and C. lusitaniae isolates. Fluconazole resistance was present in 23.52% of C. glabrata, 5.08% of C. parapsilosis and in none of the C. albicans, C. tropicalis and C. lusitaniae isolates. Echinocandin resistance was present only in 1(5.88%) C. glabrata isolate. The study emphasises the importance of monitoring local epidemiologic data and antifungal susceptibility trends due to the diversity of affected patient groups in our hospital.

Fast further purification of diastereomeric salts of a nonracemic acid by gas antisolvent fractionation.

A novel, green possibility of the further purification of the diastereomeric salt of 4-chloromandelic acid and 1-phenylethane-1-amine was developed. Gas antisolvent method using supercritical carbon dioxide was applied for the first time to precipitate the diastereomeric salts with increased purity followed by the supercritical fluid extraction of the dissolved diastereomers. The RR-salt can be purified to >99%, while fractionation-based purification of the SR-salt is limited to ~80%. The limiting initial diastereomeric excess correlates strongly with the atmospheric melting eutectic composition of the same salts, which suggests that despite the fast precipitation, the diastereomeric excess of the solid product is not kinetically determined. The efficiency of the diastereomeric enrichment is in the same range as that of the atmospheric reference experiments; however, technological advantages provided by the antisolvent precipitation method such as fast processing and dry product obtained suggest that this novel procedure is a promising alternative to the atmospheric methods.

Analysis of Romanian Bacteroides isolates for antibiotic resistance levels and the corresponding antibiotic resistance genes.

As part of an ESCMID Study Group on Anaerobic Infections (ESGAI) project, a study was conducted to measure the antibiotic susceptibilities and corresponding gene contents of 53 Bacteroides fragilis group strains isolated in Romania. The antibiotic resistance data was comparable with the data found for other East-European countries. Here, no resistant isolate was found for imipenem, metronidazole and tigecycline. An increasing role of the cepA, cfxA and cfiA genes was observed in their corresponding antibiotic resistances. Moreover, no isolate was found that harbored the cfiA gene with a possible activating IS element. Clindamycin resistance was low, similarly to that the rate for the ermF gene. However, we did find some isolates with nimB, ermB, msrSA, linA, satG, tetX, tetM and bexA genes. This study was the first to provide antibiotic resistance data for clinical Bacteroides strains from Romania.

Interactions and chemical transformations of coronene inside and outside carbon nanotubes.

By exposing flat and curved carbon surfaces to coronene, a variety of van der Waals hybrid heterostructures are prepared, including coronene encapsulated in carbon nanotubes, and coronene and dicoronylene adsorbed on nanotubes or graphite via π-π interactions. The structure of the final product is determined by the temperature of the experiment and the curvature of the carbon surface. While at temperatures below and close to the sublimation point of coronene, nanotubes with suitable diameters are filled with single coronene molecules, at higher temperatures additional dimerization and oligomerization of coronene occurs on the surface of carbon nanotubes. The fact that dicoronylene and possible higher oligomers are formed at lower temperatures than expected for vapor-phase polymerization indicates the active role of the carbon surface used primarily as template. Removal of adsorbed species from the nanotube surface is of utmost importance for reliable characterization of encapsulated molecules: it is demonstrated that the green fluorescence attributed previously to encapsulated coronene is instead caused by dicoronylene adsorbed on the surface which can be solubilized and removed using surfactants. After removing most of the adsorbed layer, a combination of Raman spectroscopy and transmission electron microscopy was employed to follow the transformation dynamics of coronene molecules inside nanotubes.

An abnormal N-heterocyclic carbene-carbon dioxide adduct from imidazolium acetate ionic liquids: the importance of basicity.

In the reaction of 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc] ionic liquid with carbon dioxide at 125 °C and 10 MPa, not only the known N-heterocyclic carbene (NHC)-CO2 adduct I, but also isomeric aNHC-CO2 adducts II and III were obtained. The abnormal NHC-CO2 adducts are stabilized by the presence of the polarizing basic acetate anion, according to static DFT calculations and ab initio molecular dynamics studies. A further possible reaction pathway is facilitated by the high basicity of the system, deprotonating the initially formed NHC-CO2 adduct I, which can then be converted in the presence of the excess of CO2 to the more stable 2-deprotonated anionic abnormal NHC-CO2 adduct via the anionic imidazolium-2,4-dicarboxylate according to DFT calculations on model compounds. This suggests a generalizable pathway to abnormal NHC complex formation.

First description of bla(NDM-1), bla(OXA-48), bla(OXA-181) producing Enterobacteriaceae strains in Romania.

We report the first isolation and characterization of several Enterobacteriaceae strains harboring bla(NDM-1), bla(OXA-48) and/or bla(OXA-181) genes in a Romanian emergency teaching hospital. Between January 2010 and September 2012 nine carbapenemase-producing Enterobacteriaceae strains were identified. The bla(NDM-1) gene was present in two Enterobacter cloacae strains, an Escherichia coli and two Klebsiella pneumoniae strains. One of these K. pneumoniae strains also harbored the bla(OXA-181) gene. Three other K. pneumoniae strains and one Serratia marcescens carried bla(OXA-48).

Continuous Stripping with Dense Carbon Dioxide.

The integration of flow chemistry into continuous manufacturing requires efficient, controllable, and continuous methods for the concentration of diluted solutions on relatively small scales. The design and application examples of a new continuous solvent removal process are presented. The continuous stripping method employing dense carbon dioxide is based on the formation of homogeneous mixtures of dilute organic solutions of the target molecules with a large excess of carbon dioxide at temperatures as low as 35 °C and pressures around 10 MPa. Subsequent pressure reduction results in the quick release of carbon dioxide and vaporization of a significant fraction of the organic solvent. The concentration of the solute in the separated liquid phase can be up to 40 times higher than in the feed. Among the many controllable process parameters, the most significant ones are the mass-flow rate ratio of carbon dioxide to the feed and the temperature of the phase separator. By careful setting of the operational parameters, the degree of concentration enhancement may be accurately controlled. The new apparatus-despite consisting of laboratory equipment and being built in a fume hood-could easily support pilot-scale synthetic flow chemistry, being a continuous, efficient alternative to thermal concentration methods.

Comparison of Six Phenotypic Assays with Reference Methods for Assessing Colistin Resistance in Clinical Isolates of Carbapenemase-Producing Enterobacterales: Challenges and Opportunities.

The global escalation of severe infections due to carbapenemase-producing Enterobacterales (CPE) isolates has prompted increased usage of parenteral colistin. Considering the reported difficulties in assessing their susceptibility to colistin, the purpose of the study was to perform a comparative evaluation of six phenotypic assays-the colistin broth disc elution (CBDE), Vitek 2 Compact (bioMérieux SA, Marcy l'Etoile, France), the Micronaut MIC-Strip Colistin (Merlin Diagnostika GMBH, Bornheim-Hensel, Germany), the gradient diffusion strip Etest (bioMérieux SA, Marcy l'Etoile, France), ChromID Colistin R Agar (COLR) (bioMérieux SA, Marcy l'Etoile, France), and the Rapid Polymyxin NP Test (ELITechGroup, Signes, France)-versus the reference method of broth microdilution (BMD). All false resistance results were further assessed using population analysis profiling (PAP). Ninety-two nonrepetitive clinical CPE strains collected from two hospitals were evaluated. The BMD confirmed 36 (39.13%) isolates susceptible to colistin. According to the BMD, the Micronaut MIC-Strip Colistin, the CBDE, and the COLR medium exhibited category agreement (CA) of 100%. In comparison with the BMD, the highest very major discrepancy (VMD) was noted for Etest (n = 15), and the only false resistance results were recorded for the Rapid Polymyxin NP Test (n = 3). Only the PAP method and the Rapid Polymyxin NP Test were able to detect heteroresistant isolates (n = 2). Thus, there is an urgent need to further optimize the diagnosis strategies for colistin resistance.

Characterization of Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Romanian Hospitals Co-Presenting Resistance and Heteroresistance to Colistin.

Klebsiella pneumoniae is a notorious human pathogen involved in healthcare-associated infections. The worldwide expansion of infections induced by colistin-resistant and carbapenemase-producing Enterobacterales (CPE) isolates has been increasingly reported. This study aims to analyze the phenotypic and molecular profiles of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR) (parental and the corresponding resistant mutants) isolates of K. pneumoniae CPE sourced from two hospitals. The phenotypes of strains in the selected collection had been previously characterized. Antimicrobial susceptibility testing was performed using a Vitek 2 Compact system (BioMérieux SA, Marcy l'Etoile, France), the disc diffusion method, and broth microdilution (BMD) for colistin. Whole-genome sequencing (WGS) did not uncover evidence of any mobile colistin resistance (mcr) genes, although the mgrB gene of seven isolates appeared to be disrupted by insertion sequences (ISKpn25 or ISKpn26). Possible deleterious missense mutations were found in phoP (L4F), phoQ (Q426L, L26Q, L224Q, Q317K), pmrB (R256G, P95L, T157P, V352E), and crrB (P151S) genes. The identified isolates belonged to the following clonal lineages: ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1). All strains harbored IncF plasmids. OXA-48 producers carried IncL and IncR plasmids, while one blaNDM-1 genome was found to harbor IncC plasmids. Ceftazidime-avibactam remains a therapeutic option for KPC-2 and OXA-48 producers. Resistance to meropenem-vaborbactam has emerged in some blakPC-2-carrying isolates. Our study demonstrates that the results of WGS can provide essential evidence for the surveillance of antimicrobial resistance.

New Formula for the Hydrogen-Bonding Hansen Component of Methanol, Ethanol, and n-Propanol for Non-ambient Conditions-Application in Gas Antisolvent Fractionation-Based Optical Resolution.

Optical resolution by diastereomeric salt formation based on gas antisolvent fractionation is influenced by the chemical equilibrium of the salt formation, the solubility, and the extraction of the compounds. Selectivity, also known as resolution efficiency, is highly solvent-dependent and is also affected by process parameters both in atmospheric and supercritical processes. For the first time in the literature, a mathematical model that employs all three Hansen parameters and operating parameters is constructed to describe the selectivity of a gas antisolvent fractionation process. The satisfying goodness of fit of the models suggests that the outcome of the three subprocesses in the gas antisolvent fractionation process (i.e., salt formation reaction, precipitation, and extraction) can be described in a single model. A new formula for pressure and temperature correction of the hydrogen-bonding component of the Hansen parameter for non-ambient conditions for liquid methanol, ethanol, and n-propanol is also suggested in this paper.

[Effects of bioactive molecules of Beta vulgaris L. ssp. esculenta var. rubra on metastatic prostate cancer]. / A Beta vulgaris L. ssp. esculenta var. rubra bioaktív vegyületeinek hatása metasztatikus prosztatarákban.

UNLABELLED: Several reports are known about the effects of nutrition supplements in the improvement of quality of life of patients with tumor, however, the physiological background remains largely unknown. Table beet affects numerous biochemical reactions, enzymes and metabolic-synthesis. METHODS: Natural table beet product come from commercial service was given twice 10 g daily for 1 month for 24 patients (mean age 68+/-8 years) with hormone-resistant and metastatic prostate cancer treated with taxan chemotherapy, who report themselves first, mean 3,6+/-2,8 years ago with their complains. 18 men's data were amenable after treatment for evaluation. In addition to routine laboratory examination values of HbA1c, 9 cytokines and levels of 3 growth factors, the global parameters of redox-homeostasis, few elements of their metal-ions, Zn- and level of free protoporfirin, trans-metilating processes before and 1 month after treatment were determined. RESULTS: In most of the patients, favorable impact of beet was enforced and significantly high levels of Zn- and free protoporfirin decreased; furthermore, trans-metilating processes fastened. CONCLUSIONS: According to results, it seems that moderate and permanent consumption of table beet product affects the life expectancy of patients favorably; however, due to the increasing values of EGF, medical control is necessary for patients with prostate cancer treated by chemotherapy.

[Antimicrobial use and its correlations with the frequency of carbapenem-resistant Pseudomonas aeruginosa strains in a hospital setting]. / Evaluarea consumului de antibiotice si a efectului acestuia asupra frecventei tulpinilor de Pseudomonas aeruginosa carbapenem rezistente în mediul spitalicesc.

One of the major causes leading to increased antibiotic resistance is excess antimicrobial consumption. We have analysed the correlation between antibiotic use and frequency of carbapenem-resistant Pseudomonas aeruginosa (CRPA) strains in the general intensive care unit of a university hospital Antibiotic use was expressed as number DDD (daily defined doses)/100 occupied hospital bed-days (BD). CRPA incidence rates were determined by number of unique isolates reported to 100,000 BD. The average use of antibiotics between January and August 2008 was 174 DDD/100 BD. The first four most frequently prescribed antibiotics were as follows: first and second generation cephalosporins (47 DDD/100 BD), carbapenemns (29 DDD/100 BD), fluoroquinolones (26 DDD/100 BD) and glycopeptids (20 DDD/100 BD). Average monthly incidence rate of CRPA was 546/100,000 BD (319-773/100,000 BD. CI 95%). There was a correlation between CRPA incidence rate and carbapenem plus fluoroquinolone use (Pearson coefficient of correlation r = 0.7, p < 0.05). Our data showed that the evolution of CRPA incidence rates was related to carbapenem and fluoroquinolone use.

Kinetic Modeling of a Consecutive Enzyme-Catalyzed Enantioselective Reaction in Supercritical Media.

Based on experimental data of both batch and continuous enzyme-catalyzed kinetic resolutions of (±)-trans-1,2-cyclohexanediol in supercritical carbon dioxide, kinetic models of increasing complexity were developed to explore the strengths and drawbacks of various modeling approaches. The simplest, first-order model proved to be a good fit for the batch experimental data in regions of high reagent concentrations but failed elsewhere. A more complex system that closely follows the true mechanism was able to fit the full range of experimental data, find constant reaction rate coefficients, and was successfully used to predict the results of the same reaction run continuously in a packed bed reactor. Care must be taken when working with such models, however, to avoid problems of overfitting; a more complex model is not always more accurate. This work may serve as an example for more rigorous reaction modeling and reactor design in the future.

Influence of benzylamine on the resolution of ibuprofen with (+)-(R)-phenylethylamine via supercritical fluid extraction.

The resolution of racemic ibuprofen was studied by partial diastereomer salt formation. The resolution was performed via two methods: resolution with (+)-(R)-phenylethylamine as chiral agent and resolution with a mixture of (+)-(R)-phenylethylamine and benzylamine. The diastereomers and unreacted enantiomers were separated by supercritical fluid extraction with carbon dioxide at 15 MPa and 33 degrees C. The influence of the achiral benzylamine on the resolution efficiency was studied by varying the concentrations of the structurally related amines in their mixtures, keeping the sum molar ratio of the amines to racemic ibuprofen constant at 0.55 +/- 0.02. The presence of benzylamine positively influenced the resolution efficiency at certain concentrations. The crystal structure of the salts of (+)-(R)-phenylethylamine with (-)-(R)-ibuprofen and (+)-(S)-ibuprofen, respectively, as well as the cocrystal of the benzylamine-ibuprofen salt with neutral ibuprofen molecules are presented. These structures were determined by single crystal X-ray diffraction, proving the significantly different stoichiometry of the related amines with the chiral acid, in accordance with mass balance calculations.

Genetic Features of mcr-1 Mediated Colistin Resistance in CMY-2-Producing Escherichia coli From Romanian Poultry.

Colistin is a last resort antibiotic used for the treatment of human infections associated with carbapenemase-producing Enterobacteriales. Here, we evaluated the occurrence of mcr-1 and -2 plasmid-mediated colistin resistance in colistin and/or carbapenem resistant human clinical Enterobacteriales and other gram-negative bacteria (n = 543) as well as third generation cephalosporin-resistant (3GCR) Escherichia coli isolates from poultry abattoir workers (n = 15) and poultry fecal samples (n = 92) collected from two geographically separate abattoirs in Romania. which revealed that mcr-1 was present within four sequence types (STs): ST744 (n = 7), ST57 (n = 7), ST156 (n = 2), and ST10 (n = 1). Within STs, serotypes were conserved and, notably, all except one of the mcr-1-positive isolates were found to exhibit fluoroquinolone-resistance (FQR) associated SNPs in both gyrA and parC. While there were variations in genotypes, all isolates belonging to ST744, ST57, and ST156 were rich in resistance determinants, carrying aminoglycoside-modifying enzymes genes, sulfonamide resistance gene bla TEM- 1 as well as bla CMY- 2 AmpC ß-lactamase resistance genes. They also exhibited high similarity in carriage of virulence genes; ST10, however, only carried the mcr-1 gene. Whole genome sequencing (WGS) analysis also revealed that although the mcr-1 gene was identified in a diverse population of E. coli, two STs (ST57 and ST744) predominated and interestingly, were found in isolates across both abattoirs providing evidence for clonal transmission. Also, two main genomic contexts of mcr-1 isolates were revealed with all ST57 isolates harboring the mcr-1 gene between two copies of ISApl1 (or the Tn6330 transposon) whilst a common mcr-1 containing scaffold, highly similar to IncX type mcr-1-bearing plasmids (pWI2-mcr, Accession number: LT838201), was present among mcr-1 isolates of varying phylogenetic backgrounds (ST10, ST744 and ST156). The high prevalence of the mcr-1 gene in poultry E. coli isolates with co-resistance to cephalosporins and quinolones, in a country where antimicrobial use in food production species is poorly regulated, is concerning and the findings from this study should lead to better surveillance of antimicrobial resistance (AMR) in food-production animals in Romania.

[Bacterial etiology of the conditions associated with bacteremia/septicemia during the years 2006-2007]. / Etiologia bacteriana a afectiunilor asociate cu bacteriemie/septicemie in perioada 2006-2007.

PURPOSE: Evaluation of the etiology of bacterial diseases associated with bacteremia/ sepsis in the Emergency County Hospital of Tg. Mures during 2006-2007. MATERIALS AND METHODS: Blood samples of 684 patients hospitalized in Emergency County Hospital of Tg. Mures during January 2006 - December 2007 were analyzed. The isolated germs have been identified according to standard methods. The blood culture results were centralized and processed in electronic databases. RESULTS: In 2006 and 2007 the frequency of identified germs from blood cultures remained unchanged: the predominant germs are coagulase-negative staphylococci, followed by enterobacteria (Klebsiella spp prevailing), S. aureus (mostly methicillin-resistant (MRSA)) and other bacteria. CONCLUSIONS: The main bacteria isolated from blood culture are staphylococci and enterobacteria. In medical sections undertaking invasive procedures, the infections with methicillin-resistant staphylococci are common.

[Cumulative antibiogram--means of following local resistance phenomena based on material gathered in the laboratory of microbiology]. / Antibiograma cumulativa--posibilitate de urmarire a fenomenelor de rezistenta locale bazata pe materialul laboratorului de bacteiologie.

The cumulative antibiogram (CA) represents a report which summarizes the susceptibility of commonly isolated microorganisms to usual antibiotics in a defined period of time. We have evaluated the local resistance data during the year 2007 by generating CA from the bacteriology laboratory's database in Mures County Emergency Clinical Hospital for 3 categories of microorganisms: Gram-positive cocci, Gram-negative enteric rods and non-fermenters. A total of 1382 individual antibiotic susceptibility reports were analysed. Gram-positive cocci were susceptible 100% to glycopeptides and oxazolidinones. Fifty percent of S. aureus isolates were methicillin-resistant. More than 90% of S. aureus and MRSA (methicillin-resistant S. aureus) isolates were susceptible to trimethoprim-sulfamethoxazole. Gram-negative enteric rods were 100% susceptible to carbapenems. The susceptibility of Escherichia coli and Klebsiella pneumoniae to broad spectrum cephalosporins was 72% and 26%, respectively. The activity of carbapenems against Acinetobacter spp. and Pseudomonas aeruginosa was 43% and 35%, respectively. Colistin was active in 100% of the isolated nonfermenters. CA is a useful tool for evaluation of local resistance data but careful interpretation of generated data is required in order to avoid under- or overestimation of resistance.

[Significance of metal ion metabolism and oxidative stress in male patients with porphyria cutanea tarda]. / A fémion-metabolizmus és az oxidatív stressz jelentôsége porphyria cutanea tarda betegségben szenvedô férfiakban.

In the last decades, the role of oxidative stress and trace elements was proven to play an important role in the pathogenesis of more and more diseases. This is why a great importance is attributed lately to the antioxidant therapy, and lots of studies are dealing with this issue. In porphyria cutanea tarda (PCT) the biosynthesis of hem is damaged, because of the reduced activity of uroporphyrinogen-decarboxylase enzyme. The hem precursors are accumulating in blood, liver and skin. The hem precursors and porphyrin are eliminated with urine and stool. The enzyme defect is autosomal dominant. The skin symptoms are intensified by sun exposure. This is because the accumulation of uroporphyrins and heptacarboxylporphyrin in the skin causes photosensitivity, and the accumulated iron has a lipid-peroxidation effect. Besides the genetical origin, the alcohol consumption, the hepatotoxic drugs, estrogen and viral infections can also determine the development of the disease. The applied treatment is phlebotomy. In the case of PCT that appears in the field of liver damage, the accumulation of iron is responsible for the development of oxidative stress. The patient's redox homeostasis is changed, and the level of antioxidants is decreased. The redox state of liver and the effects of additional antioxidant treatment in phlebotomized PCT patients were determined by biochemical and trace element analytical methods. According to the clinical data, phlebotomy proved to be an effective treatment in PCT patients. Phlebotomy improved the phototoxic skin symptoms, but it did not improve the ratios of trace elements to each other in the blood of the patients.