Phenotypic and molecular characterizations of carbapenem-resistant Acinetobacter baumannii isolates collected within the EURECA study.

Multi-drug-resistant Acinetobacter baumannii isolates are key pathogens that contribute to the global burden of antimicrobial resistance. This study aimed to investigate the phenotypic and molecular characteristics of carbapenem-resistant A. baumannii (CRAB) isolates from the EURECA clinical trial. In total, 228 CRAB clinical strains were recovered from 29 sites in 10 European countries participating in the EURECA study between May 2016 and November 2018. All strains were reconfirmed centrally for identification and antimicrobial susceptibility testing, and were then subjected to DNA isolation and whole-genome sequencing (WGS), with analysis performed using BacPipe v.1.2.6. K and O typing was performed using KAPTIVE. Overall, 226 (99.1%) strains were confirmed as CRAB isolates. The minimum inhibitory concentration (MIC90) results of imipenem and meropenem were >16 mg/L. WGS showed that the isolates mainly harboured blaOXA-23 (n=153, 67.7%) or blaOXA-72 (n=70, 30.1%). Four blaOXA-72 isolates from Serbia co-harboured blaNDM-1. An IS5 transposase family element, ISAba31, was found upstream of the blaOXA-72 gene harboured on a small (~10-kb) pSE41030-EUR plasmid. The majority of isolates (n=178, 79.1%) belonged to international clone II. Strains belonging to the same sequence type but isolated in different countries or within the same country could be delineated in different clusters by core-genome multi-locus sequence typing (MLST). Whole-genome/core-genome MLST showed high diversity among the isolates, and the most common sequence type was ST2 (n=153, 67.7%). The EURECA A. baumannii strain collection represents a unique, diverse repository of carbapenem-resistant isolates that adds to the existing knowledge of A. baumannii epidemiology and resistance genes harboured by these strains.

Review and in silico analysis of fermentation, bioenergy, fiber, and biopolymer genes of biotechnological interest in Agave L. for genetic improvement and biocatalysis.

Several of the over 200 known species of Agave L. are currently used for production of distilled beverages and biopolymers. The plants live in a wide range of stressful environments as a result of their resistance to abiotic stress (drought, salinity, and extreme temperature) and pathogens, which gives the genus potential for germplasm conservation and biotechnological applications that may minimize economic losses as a result of the global climate change. However, the limited knowledge in the genus of genome structure and organization hampers development of potential improved biotechnological applications by means of genetic manipulation and biocatalysis. We reviewed Agave and plant sequences in the GenBank NCBI database for identifying genes with biotechnological potential for fermentation, bioenergy, fiber improvement, and in vivo plant biopolymer production. Three-dimensional modeling of enzyme structures in plant accessions revealed structural differences in sucrose 1-fructosyltransferase, fructan 1-fructosyltransferase, fructan exohydrolase (1-FEH), cellulose synthase (CES), and glucanases (EGases) with possible effects in fructan, sugar, and biopolymer production. Although the coding genes of FEH and enzymes involved in biopolymer production (CES, sucrose synthase, and EGases) remain unidentified in Agave L., our results could aid isolation of such genes in Agave. By comparing nucleotide and amino acid sequences in accessions of Agave and other plants, knowledge may be gained about transcriptional regulation and enzymatic activity factors. Future study is needed of biotechnological application of Agave genes for crop breeding aided by genetic engineering and biocatalysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1314-1334, 2018.

Comparative in vitro activity of oral antimicrobial agents against Enterobacteriaceae from patients with community-acquired urinary tract infections in three European countries.

Enterobacteriaceae causing community-acquired urinary tract infections were examined in selected outpatient clinics and hospitals in Belgium, Germany and Spain using EUCAST breakpoints for susceptibility. A total of 1190 isolates were collected. Escherichia coli isolates were resistant to amoxicillin-clavulanic acid (28.1%), ciprofloxacin (23.4%) and trimethoprim-sulfamethoxazole (21.4%) compared with fosfomycin and nitrofurantoin (each, <1.5%). Ceftibuten (MIC50/90 0.25/0.5 mg/L) and ceftriaxone activity (MIC50/90 ≤0.25 mg/L) was comparable. Ceftibuten (MIC90 ≤0.25 mg/L) was also active against Proteus mirabilis and Klebsiella spp. Extended-spectrum ß-lactamase phenotypes were 7.1% for E. coli, 5.6% for Klebsiella pneumoniae and 0.4% for P. mirabilis. Resistance was common among men and elderly women.

Assessment of the Phoenix™ automated system and EUCAST breakpoints for antimicrobial susceptibility testing against isolates expressing clinically relevant resistance mechanisms.

EUCAST breakpoint criteria are being adopted by automatic antimicrobial susceptibility testing systems. The accuracy of the Phoenix Automated System in combination with 2012 EUCAST breakpoints against recent clinical isolates was evaluated. A total of 697 isolates (349 Enterobacteriaceae, 113 Pseudomonas spp., 25 Acinetobacter baumannii, 11 Stenotrophomonas maltophilia, 95 Staphylococcus aureus, 6 coagulase negative staphylococci, 77 enterococci and 21 Streptococcus pneumoniae) with defined resistance phenotypes and well-characterized resistance mechanisms recovered in Spain (n = 343) and Italy (n = 354) were tested. Comparator antimicrobial susceptibility testing data were obtained following CLSI guidelines. Experimental agreement (EA), defined as MIC agreement ±1 log(2) dilution, category agreement (CA) and relative discrepancies (minor (mD), major (MD) and very major discrepancies (VMD)) were determined. The overall EA and CA for all organism-antimicrobial agent combinations (n = 6.294) were 97.3% and 95.2%, respectively. mD, MD and VMD were 4.7%, 1.3% and 2.7%, all of them in agreement with the ISO (ISO20776-2:2007) acceptance criteria for assessment of susceptibility testing devices. VMD were mainly observed in amoxicillin-clavulanate and cefuroxime in Enterobacteriaceae and gentamicin in Pseudomonas aeruginosa, whereas MD were mainly observed in amoxicillin-clavulante in Enterobacteriaceae. mD were mainly observed in Enterobacteriaceae but distributed in different antimicrobials. For S. aureus and enterococci relative discrepancies were low. The Phoenix system showed accuracy assessment in accordance with the ISO standards when using EUCAST breakpoints. Inclusion of EUCAST criteria in automatic antimicrobial susceptibility testing systems will facilitate the implementation of EUCAST breakpoints in clinical microbiology laboratories.

Clinical and microbiological features of a cystic fibrosis patient chronically colonized with Pandoraea sputorum identified by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Clonal isolates identified as various nonfermentative Gram-negative bacilli over a 5-year period from sputum cultures of a 30-year-old cystic fibrosis patient were successfully reidentified as Pandoraea sputorum by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Decreased lung function improved after 1 year of azithromycin and inhaled 7%-hypertonic saline treatment.

Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients.

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade-offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well-defined collection of 42 CF Pseudomonas aeruginosa isolates. MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10(-8)) than in biofilm (9.9 × 10(-9)) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10(-9) in the planktonic mode to 6 × 10(-8) in biofilm, whereas normomutators (from 9.4 × 10(-8) to 5.3 × 10(-8)) and hypermutators (from 1.6 × 10(-6) to 7.7 × 10(-7)) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa, leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.

Characterization of flavocytochrome C552 from the thermophilic photosynthetic bacterium Chromatium tepidum.

A M(r) 68 kDa flavocytochrome c552 has been isolated from the thermophilic photosynthetic purple sulfur bacterium Chromatium tepidum and shown to consist of a M(r) 25 kDa subunit that contains two covalently bound heme c and a M(r) 43 kDa subunit that probably contains a single FAD. The prosthetic group content, absorbance spectra, and subunit composition of the C. tepidum flavocytochrome are quite similar to those previously reported for the flavocytochrome c552 isolated from a mesophilic Chromatium species, Chromatium vinosum. The oxidation-reduction properties of the hemes present in the C. tepidum flavocytochrome have been characterized by titrations, the effect of temperature on the catalytic activity of the protein has been investigated, and the heme environment has been characterized using resonance Raman spectroscopy.

Spectroscopic characterization of flavocytochrome c-552 from the photosynthetic purple sulfur bacterium Chromatium vinosum.

The identities of the axial ligands to the two hemes of the flavocytochrome c-552 isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum have been investigated by visible/near-infrared absorption and magnetic circular dichroism (MCD) spectroscopies, with parallel electron paramagnetic resonance (EPR) studies. One of the hemes has histidine and methionine as axial ligands and has a local environment that is relatively insensitive to the composition of the bulk medium. The second heme, the local environment of which is sensitive to changes in the composition of the bulk medium, exists as a mixture of two forms, only one of which has histidine/methionine axial ligation. On the basis of its EPR characteristics, the other form most likely has histidine/lysine axial ligation. In aqueous solution near neutral pH, more than half of the second heme is present as the histidine/lysine form, while in 50:50 water/ethylene glycol the histidine/methionine form is the dominant one.