Sliding motility of Bacillus cereus mediates vancomycin pseudo-resistance during antimicrobial susceptibility testing.

BACKGROUND: The glycopeptide vancomycin is the antimicrobial agent-of-choice for the treatment of severe non-gastrointestinal infections with members of Bacillus cereus sensu lato (s.l.). Recently, sporadic detection of vancomycin-resistant phenotypes emerged, mostly for agar diffusion testing such as the disc diffusion method or gradient test (e.g. Etest®) method. RESULTS: In this work, we were able to disprove a preliminarily assumed high resistance to vancomycin in an isolate of B. cereus s.l. using broth microdilution and agar dilution. Microscopic imaging during vancomycin susceptibility testing showed spreading towards the inhibition zone, which strongly suggested sliding motility. Furthermore, transcriptomic analysis using RNA-Seq on the nanopore platform revealed several key genes of biofilm formation (e.g. calY, tasA, krsEABC) to be up-regulated in pseudo-resistant cells, substantiating that bacterial sliding is responsible for the observed mobility. Down-regulation of virulence (e.g. hblABCD, nheABC, plcR) and flagellar genes compared with swarming cells also confirmed the non-swarming phenotype of the pseudo-resistant isolate. CONCLUSIONS: The results highlight an insufficiency of agar diffusion testing for vancomycin susceptibility in the B. cereus group, and reference methods like broth microdilution are strongly recommended. As currently no guideline mentions interfering phenotypes in antimicrobial susceptibility testing of B. cereus s.l., this knowledge is essential to obtain reliable results on vancomycin susceptibility. In addition, this is the first report of sliding motility undermining accurate antimicrobial susceptibility testing in B. cereus s.l. and may serve as a basis for future studies on bacterial motility in susceptibility testing and its potential impact on treatment efficacy.

Fiber-based food packaging materials in view of bacterial growth and survival capacities.

Understanding interactions of bacteria with fiber-based packaging materials is fundamental for appropriate food packaging. We propose a laboratory model to evaluate microbial growth and survival in liquid media solely consisting of packaging materials with different fiber types. We evaluated food contaminating species (Escherichia coli, Staphylococcus aureus, Bacillus cereus), two packaging material isolates and bacterial endospores for their growth abilities. Growth capacities differed substantially between the samples as well as between bacterial strains. Growth and survival were strongest for the packaging material entirely made of recycled fibers (secondary food packaging) with up to 10.8 log10 CFU/ml for the packaging isolates. Among the food contaminating species, B. cereus and E. coli could grow in the sample of entirely recycled fibers with maxima of 6.1 log10 and 8.6 log10 CFU/mL, respectively. Escherichia coli was the only species that was able to grow in bleached fresh fibers up to 7.0 log10 CFU/mL. Staphylococcus aureus perished in all samples and was undetectable after 1-6 days after inoculation, depending on the sample. The packaging material strains were isolated from recycled fibers and could grow only in samples containing recycled fibers, indicating an adaption to this environment. Spores germinated only in the completely recycled sample. Additionally, microbial digestion of cellulose and xylan might not be a crucial factor for growth. This is the first study describing bacterial growth in food packaging materials itself and proposing functionalization strategies toward active food packaging through pH-lowering.

Modelling and Determination of Parameters Influencing the Transfer of Microorganisms from Food Contact Materials.

The transfer of microorganisms on packaging materials to a contact surface has only been investigated in the context of laboratory-produced spiked packaging products and agar surfaces in small quantities (0.03-0.10%) so far. Correspondingly, this study focused on the localization of microorganisms on/in industrially produced packaging materials and on the establishment of an experimental laboratory set-up to determine and quantify the parameters influencing the microbial transport from surfaces and different layers of packaging materials to contact agar media. We established a simple model to determine the transfer of microorganisms from packaging materials to microbiological agar plates. In order to clarify the transfer of microorganisms within the material, the samples were split horizontally in their z-dimension, and so produced layers (inner layers) were investigated for their microbial transfer. The parameters incubation time, applied weight and bacterial load for the samples were investigated in more detail in the outer layers (front/back) and the inner layers. No significant difference in the microbial transfer was observed between the outer and inner layers of all samples. We indicate a time-dependent transfer to the media and an independence of the transfer from the applied weight. Moreover, the number of transferred microorganisms is not dependent on the bacterial load of the samples.

Excitation localization in a trimeric perylenediimide macrocycle: Synthesis, theory, and single molecule spectroscopy.

A novel trimeric perylenediimide (PDI) macrocycle was synthesized, and its intramolecular electronic couplings were investigated by bulk and single-molecule optical spectroscopy and by various theoretical approaches. In polarization-resolved excitation spectroscopy at 1.2 K in a PMMA matrix, the appearance and disappearance of the three zero-phonon lines (ZPLs) of an individual trimer by changing the polarization in steps of 60° nicely reflect an approximate triangular geometry of the macrocycle and indicate localized excitations that are transferred by incoherent hopping processes at time scales of around 1 ps as inferred from the ZPL linewidths. The electronic coupling strength deduced from the low temperature data is found to be in good agreement with theoretical estimates. Bulk spectroscopy in toluene at room temperature indicates that the excitations are also localized under these conditions. Theory reveals that the reasons for the localized nature of the excitations at room and low temperatures are different. For a rigid macrocycle, the excitations are predicted to be delocalized, but molecular dynamics simulations point to considerable structural flexibility at ambient temperatures, which counteracts excitation delocalization. At 1.2 K in a PMMA matrix, this effect is too small to lead to localization. Yet, supported by simple model calculations, the disorder in the PMMA host induces sufficient differences between the PDI chromophores, which again result in localized excitations. By addressing crucial aspects of excitation energy transfer, our combined approach provides a detailed and quantitative account of the interchromophore communication in a trimeric macrocycle.

Bacillus cereus in Packaging Material: Molecular and Phenotypical Diversity Revealed.

The Bacillus cereus group has been isolated from soils, water, plants and numerous food products. These species can produce a variety of toxins including several enterotoxins [non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), cytotoxin K, and enterotoxin FM], the emetic toxin cereulide and insecticidal Bt toxins. This is the first study evaluating the presence of B. cereus in packaging material. Among 75 different isolates, four phylogenetic groups were detected (II, III, IV, and VI), of which the groups III and IV were the most abundant with 46.7 and 41.3%, respectively. One isolate was affiliated to psychrotolerant group VI. Growth experiments showed a mesophilic predominance. Based on PCR analysis, nhe genes were detectable in 100% of the isolates, while hbl genes were only found in 50.7%. The cereulide encoding gene was found in four out of 75 isolates, no isolate carried a crystal toxin gene. In total, thirteen different toxin gene profiles were identified. We showed that a variety of B. cereus group strains can be found in packaging material. Here, this variety lies in the presence of four phylogenetic groups, thirteen toxin gene profiles, and different growth temperatures. The results suggest that packaging material does not contain significant amounts of highly virulent strains, and the low number of cereulide producing strains is in accordance with other results.

Twisting versus Delocalization in CAAC- and NHC-Stabilized Boron-Based Biradicals: The Roles of Sterics and Electronics.

Invited for the cover of this issue is Bernd Engels, Holger Braunschweig, Volker Engel and their coworkers at University of Würzburg. The image depicts bridged boron compounds which possess fascinating relationships between their composition and their geometrical and electronic structures, the latter ranging from closed-shell to biradical triplet or singlet ground state. Read the full text of the article at 10.1002/chem.202004619.

Twisting versus Delocalization in CAAC- and NHC-Stabilized Boron-Based Biradicals: The Roles of Sterics and Electronics.

Twisted boron-based biradicals featuring unsaturated C2 R2 (R=Et, Me) bridges and stabilization by cyclic (alkyl)(amino)carbenes (CAACs) were recently prepared. These species show remarkable geometrical and electronic differences with respect to their unbridged counterparts. Herein, a thorough computational investigation on the origin of their distinct electrostructural properties is performed. It is shown that steric effects are mostly responsible for the preference for twisted over planar structures. The ground-state multiplicity of the twisted structure is modulated by the σ framework of the bridge, and different R groups lead to distinct multiplicities. In line with the experimental data, a planar structure driven by delocalization effects is observed as global minimum for R=H. The synthetic elusiveness of C2 R2 -bridged systems featuring N-heterocyclic carbenes (NHCs) was also investigated. These results could contribute to the engineering of novel main group biradicals.

cAAC-Stabilized 9,10-diboraanthracenes-Acenes with Open-Shell Singlet Biradical Ground States.

Narrow HOMO-LUMO gaps and high charge-carrier mobilities make larger acenes potentially high-efficient materials for organic electronic applications. The performance of such molecules was shown to significantly increase with increasing number of fused benzene rings. Bulk quantities, however, can only be obtained reliably for acenes up to heptacene. Theoretically, (oligo)acenes and (poly)acenes are predicted to have open-shell singlet biradical and polyradical ground states, respectively, for which experimental evidence is still scarce. We have now been able to dramatically lower the HOMO-LUMO gap of acenes without the necessity of unfavorable elongation of their conjugated π system, by incorporating two boron atoms into the anthracene skeleton. Stabilizing the boron centers with cyclic (alkyl)(amino)carbenes gives neutral 9,10-diboraanthracenes, which are shown to feature disjointed, open-shell singlet biradical ground states.

Naphthoquinones as Covalent Reversible Inhibitors of Cysteine Proteases-Studies on Inhibition Mechanism and Kinetics.

The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action.

Lewis-Base Stabilization of the Parent Al(I) Hydride under Ambient Conditions.

Aluminum(III) is inherently electron deficient and therefore acts as a prototypical Lewis acid. Conversely, Al(I) is a rare, nucleophilic variant of aluminum that is thermodynamically unstable under ambient conditions. While attempts to stabilize and isolate Al(I) species have become increasingly successful, the parent Al(I) (i.e, Al-H) remains accessible only under extreme temperatures/pressures or matrix conditions. Here, we report the isolation of the parent Al(I) hydride under ambient conditions via the reduction of a Lewis-base-stabilized alkyldihaloalane. Computational and spectroscopic analyses indicate that the ground-state electronic configuration of this monomeric aluminum species is best described as an Al(I) hydride with non-negligible open-shell Al(III) singlet diradical character. These findings are also supported by reactivity studies, which reveal both the p-centered lone pair donating ability and the hydridic nature of the parent aluminene.

Catalyst-free room-temperature iClick reaction of molybdenum(ii) and tungsten(ii) azide complexes with electron-poor alkynes: structural preferences and kinetic studies.

Two isostructural and isoelectronic group VI azide complexes of the general formula [M(η3-allyl)(N3)(bpy)(CO)2] with M = Mo, W and bpy = 2,2'-bipyridine were prepared and fully characterized, including X-ray structure analysis. Both reacted smoothly with electron-poor alkynes such as dimethyl acetylenedicarboxylate (DMAD) and 4,4,4-trifluoro-2-butynoic acid ethyl ester in a catalyst-free room-temperature iClick [3 + 2] cycloaddition reaction. Reaction with phenyl(trifluoromethyl)acetylene, on the other hand, did not lead to any product formation. X-ray structures of the four triazolate complexes isolated showed the monodentate ligand to be N2-coordinated in all cases, which requires a 1,2-shift of the nitrogen from the terminal azide to the triazolate cycloaddition product. On the other hand, a 19F NMR spectroscopic study of the reaction of the fluorinated alkyne with the tungsten azide complex at 27 °C allowed detection of the N1-coordinated intermediate. With this method, the second-order rate constant was determined as (7.3 ± 0.1) × 10-2 M-1 s-1, which compares favorably with that of first-generation compounds such as difluorocyclooctyne (DIFO) used in the strain-promoted azide-alkyne cycloaddition (SPAAC). In contrast, the reaction of the molybdenum analogue was too fast to be studied with NMR methods. Alternatively, solution IR studies revealed pseudo-first order rate constants of 0.4 to 6.5 × 10-3 s-1, which increased in the order of Mo > W and F3C-C[triple bond, length as m-dash]C-COOEt > DMAD.

A UGT2B10 splicing polymorphism common in african populations may greatly increase drug exposure.

RO5263397 [(S)-4-(3-fluoro-2-methyl-phenyl)-4,5-dihydro-oxazol-2-ylamine], a new compound that showed promising results in animal models of schizophrenia, is mainly metabolized in humans by N-glucuronidation. Enzyme studies, using the (then) available commercial uridine 5'-diphosphate-glucuronosyltransferases (UGTs), suggested that UGT1A4 is responsible for its conjugation. In the first clinical trial, in which RO5263397 was administered orally to healthy human volunteers, a 136-fold above-average systemic exposure to the parent compound was found in one of the participants. Further administration in this trial identified two more such poor metabolizers, all three of African origin. Additional in vitro studies with recombinant UGTs showed that the contribution of UGT2B10 to RO5263397 glucuronidation is much higher than UGT1A4 at clinically relevant concentrations. DNA sequencing in all of these poor metabolizers identified a previously uncharacterized splice site mutation that prevents assembly of full-length UGT2B10 mRNA and thus functional UGT2B10 protein expression. Further DNA database analyses revealed the UGT2B10 splice site mutation to be highly frequent in individuals of African origin (45%), moderately frequent in Asians (8%) and almost unrepresented in Caucasians (<1%). A prospective study using hepatocytes from 20 individual African donors demonstrated a >100-fold lower intrinsic clearance of RO5263397 in cells homozygous for the splice site variant allele. Our results highlight the need to include UGT2B10 when screening the human UGTs for the enzymes involved in the glucuronidation of a new compound, particularly when there is a possibility of N-glucuronidation. Moreover, this study demonstrates the importance of considering different ethnicities during drug development.