MALDI Research Articles

Identification of Enterococcus spp. from food sources by matrix-assisted laser desorption ionization-time of flight mass spectrometry and characterization of virulence factors, antibiotic resistance, and biofilm formation

The continuous detection of multi-drug-resistant enterococci in food source environments has aroused widespread concern. In this study, 198 samples from chicken products, animal feces, raw milk, and vegetables were collected in Japan and Egypt to investigate the prevalence of enterococci and virulence characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for species identification and taxonomic analysis of the isolates. The results showed that the rates of most virulence genes (efaA, gelE, asa1, ace, and hyl) in the Japanese isolates were slightly higher than those in the Egyptian isolates. The rate of efaA was the highest (94.9 %) among seven virulence genes detected, but the cylA gene was not detected in all isolates, which was in accordance with γ-type hemolysis phenotype. In Enterococcus faecalis, the rate of kanamycin-resistant strains was the highest (84.75 %) among the antibiotics tested. Moreover, 78 % of E. faecalis strains exhibited multi-drug resistance. Four moderately vancomycin-resistant strains were found in Egyptian isolates, but none were found in Japanese isolates. MALDI-TOF MS analysis correctly identified 98.5 % (68/69) of the Enterococcus isolates. In the principal component analysis dendrogram, strains isolated from the same region with the same virulence characteristics and similar biofilm-forming abilities were characterized by clustered distribution in different clusters. This finding highlights the potential of MALDI-TOF MS for classifying E. faecalis strains from food sources.

Matrix-assisted laser desorption/ionization matrix incorporation evaluation algorithm for improved peak coverage and signal-to-noise ratio in mass spectrometry imaging.

Despite decades of implementation, the selection of optimal sample preparation conditions for matrix-assisted laser desorption/ionization (MALDI) imaging is still ambiguous due to the lack of a universal and comprehensive evaluation methodology. Thus, numerous experiments with different matrix application conditions accompany a translation of the method to novel sample types and matrices. Mouse brain tissues were covered with 9-aminoacridine through sublimation, followed by recrystallization in vapors of 5% (v/v) methanol solution in water. The samples were analyzed by MALDI time-of-flight mass spectrometry, and the efficiency of lipid and small-molecule ionization was evaluated with different metrics. We first investigate the dependency of matrix density and recrystallization conditions on the thickness of an analyte-empty matrix layer to roughly evaluate the laser shot number required to obtain an intense signal with minimal noise. Then, we introduce metrics for the analysis of small imaging datasets (small sample regions) of model samples based on median quantity of peaks in spectra (medQP) and weighted median signal-to-noise ratio (wmSNR). The evaluation of small regions and taking median values for metrics help overcome the sample heterogeneity and allow for the simultaneous comparison of different acquisition parameters. Here, we propose a methodology based on gradual laser ablation of small regions of sample and further implementation of weighted signal-to-noise ratio to assess various matrix application conditions. The proposed approach helps reduce the number of test samples required to determine optimal sample preparation conditions and improve the overall quality of images.

Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen.

This study aimed to identify and characterize a novel endo-β-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. A novel endo-β-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley β-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40°C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley β-glucan (109.59±3.61 μmol/mg min) and Icelandic moss lichenan (35.35±1.55 μmol/mg min). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.

Development of a New Binary Matrix for the Comprehensive Analysis of Lipids and Pigments in Micro- and Macroalgae Using MALDI-ToF/ToF Mass Spectrometry.

While edible algae might seem low in fat, the lipids they contain are crucial for good health and preventing chronic diseases. This study introduces a binary matrix to analyze all the polar lipids in both macroalgae (Wakame-Undaria pinnatifida, Dulse-Palmaria palmata, and Nori-Porphyra spp.) and microalgae (Spirulina-Arthrospira platensis, and Chlorella-Chlorella vulgaris) using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The key lies in a new dual matrix made by combining equimolar amounts of 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). This combination solves the limitations of single matrices: 9AA is suitable for sulfur-containing lipids and acidic phospholipids, while DAN excels as an electron-transfer secondary reaction matrix for intact chlorophylls and their derivatives. By employing the equimolar binary matrix, a wider range of algal lipids, including free fatty acids, phospholipids, glycolipids, pigments, and even rare arsenosugarphospholipids were successfully detected, overcoming drawbacks related to ion suppression from readily ionizable lipids. The resulting mass spectra exhibited a good signal-to-noise ratio at a lower laser fluence and minimized background noise. This improvement stems from the binary matrix's ability to mitigate in-source decay effects, a phenomenon often encountered for certain matrices. Consequently, the data obtained are more reliable, facilitating a faster and more comprehensive exploration of algal lipidomes using high-throughput MALDI-MS/MS analysis.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions.

Negative-ion electron capture dissociation (niECD) is an anion MS/MS technique that provides fragmentation analogous to conventional ECD, including high peptide sequence coverage and retention of labile post-translational modifications (PTMs). niECD has been proposed to be the most efficient for salt-bridged zwitterionic precursor ion structures. Several important PTMs, e.g., sulfation and phosphorylation, are acidic and can, therefore, be challenging to characterize in the positive-ion mode. Furthermore, PTM-friendly techniques, such as ECD, require multiple precursor ion-positive charges. By contrast, singly charged ions, refractory to ECD, are most compatible with niECD. Because electrospray ionization (ESI) typically yields multiply charged ions, we sought to explore matrix-assisted laser desorption/ionization (MALDI) in combination with niECD. However, the requirement for zwitterionic gaseous structures may preclude efficient niECD of MALDI-generated anions. Unexpectedly, we found that niECD of anions from MALDI is not only possible but proceeds with similar or higher efficiency compared with ESI-generated anions. Matrix selection did not appear to have a major effect. With MALDI, niECD is demonstrated up to m/z ∼4300. For such larger analytes, multiple electron captures are observed, resulting in triply charged fragments from singly charged precursor ions. Such charge-increased fragments show improved detectability. Furthermore, significantly improved (∼20-fold signal-to-noise increase) niECD spectral quality is achieved with equivalent sample amounts from MALDI vs ESI. Overall, the reported combination with MALDI significantly boosts the analytical utility of niECD.

Sequencing of Phosphopeptides Using a Sequential Charge Inversion Ion/Ion Reaction and Electron Capture Dissociation Workflow.

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization.

Qualitative Analysis of Nitrogen and Sulfur Compounds in Vacuum Gas Oils via Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry.

Analysis of the heavy fractions in crude oil has been important in petroleum industries. It is well known that heavy fractions such as vacuum gas oils (VGOs) include heteroatoms, of which sulfur and nitrogen are often characterized in many cases. We conducted research regarding the molecular species analysis of VGOs. Further refine processes using VGOs are becoming important when considering carbon recycling. In this work, we attempted to classify compounds within VGOs provided by Kuwait Institute for Scientific Research. Two VGOs were priorly distillated from Kuwait Export crude and Lower Fars crude. Quantitative analysis was performed mainly using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOFMS). MALDI-TOF-MS has been developed for analyzing high-molecular-weight compounds such as polymer and biopolymers. As matrix selection is one of the most important aspects in MALDI-TOFMS, the careful selection of a matrix was firstly evaluated, followed by analysis using a Kendrick plot with nominal mass series (z*). The objective was to evaluate if this work could provide an effective classification of VGOs compounds. The Kendrick plot is a well-known method for processing mass data. The difference in the Kendrick mass defect (KMD) between CnH2n-14S and CnH2n-20O is only 0.0005 mass units, which makes it difficult in general to distinguish these compounds. However, since the z* value showed effective differences during the classification of these compounds, qualitative analysis could be possible. The analysis using nominal mass series showed the potential to be used as an effective method in analyzing heavy fractions.

The prevalent dynamic and genetic characterization of mcr-1 encoding multidrug resistant Escherichia coli strains recovered from poultry in Hebei, China

ObjectivesColistin is known as the last resort antibiotic to treat the infections caused by multidrug resistant foodborne pathogens. The emergence and widespread dissemination of plasmid-mediated colistin resistance gene mcr-1 in the Escherichia coli (E. coli) incurs potential threat to public health. Here, we investigated the epidemiology, transmission dynamics, and genetic characterization of mcr-1 harbouring E. coli isolates from poultry originated in Hebei Province, China. MethodsA total of 297 faecal samples were collected from the two large poultry farms in Hebei Province, China. The samples were processed for E. coli identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry and 16S rDNA sequencing. Then, the mcr-1 gene harbouring E. coli strains were identified by polymerase chain reaction and subjected to antimicrobial susceptibility testing by broth microdilution assay. The genomic characterization of the isolates was done by whole genome sequencing using the various bioinformatics tools, and multi-locus sequence typing was done by sequence analysis of the seven housekeeping genes. The conjugation experiment was done to check the transferability of mcr-1 along with the plasmid stability testing. ResultsA total of six mcr-1 E. coli isolates with minimum inhibitory concentration of 4 μg/mL were identified from 297 samples (2.02%). The mcr-1 harbouring E. coli were identified as multidrug resistant and belonged to ST101 (n = 4) and ST410 (n = 2). The genetic environment of mcr-1 presented its position on IncHI2 plasmid in 4 isolates and p0111 in 2 isolates, which is a rarely reported plasmid type for mcr-1. Moreover, both type of plasmids was transferable to recipient J53, and mcr-1 was flanked by 3 mobile elements ISApl1, Tn3, and IS26 forming a novel backbone Tn3–IS26-mcr-1– pap2-ISApl1 on the p0111 plasmid. The phylogenetic analysis shared a common lineage with mcr-1 harbouring isolates from the environment, humans, and animals, which indicate its horizontal spread among the diverse sources, species, and hosts. ConclusionThis study recommends the one health approach for future surveillance across multiple sources and bacterial species to adopt relevant measures and reduce global resistance crises.

Genomic characterization of carbapenem and colistin-resistant Klebsiella pneumoniae isolates from humans and dogs.

Carbapenem and colistin-resistant Enterobacteriaceae, including Klebsiella pneumoniae, have become a growing global concern, posing a significant threat to public health. Currently, there is limited information about the genetic background of carbapenem and colistin-resistant K. pneumoniae isolates infecting humans and dogs in Thailand. This study aimed to characterize carbapenem and colistin-resistant genes in six resistant K. pneumoniae clinical isolates (three from humans and three from dogs) which differed in their pulse field gel electrophoresis profiles. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), antimicrobial susceptibility testing, and whole-genome sequencing were employed to identify and analyze the isolates. All six isolates were carbapenemase-producing K. pneumoniae isolates with chromosomally carried blaSHV, fosA, oqxA and oqxB genes, as well as nine to 21 virulence genes. The isolates belonged to five multilocus sequence types (STs): one isolate from a human and one from a dog belonged to ST16, with the other two human isolates being from ST340 and ST1269 and the other two dog isolates were ST147 and ST15. One human isolate and two dog isolates harbored the same blaOXA-232 gene on the ColKP3 plasmid, and one dog isolate carried the blaOXA-48 gene on the IncFII plasmid. Notably, one human isolate exhibited resistance to colistin mediated by the mcr-3.5 gene carried on the IncFII plasmid, which co-existed with resistance determinants to other antibiotics, including aminoglycosides and quinolones. In conclusion, this study provides a comprehensive characterization of both chromosome- and plasmid-mediated carbapenem and colistin resistance in a set of K. pneumoniae clinical isolates from unrelated humans and dogs in Thailand. The similarities and differences found contribute to our understanding of the potential widescale dissemination of these important resistance genes among clinical isolates from humans and animals, which in turn may contribute to outbreaks of emerging resistant clones in hospital settings.

An evaluation of a Stenotrophomonas maltophilia outbreak due to commercial arterial blood gas collection kit

BackgroundStenotrophomonas maltophilia is a gram-negative bacterium that can cause hospital infections and outbreaks within hospitals. This study aimed to evaluate an outbreak of Stenotrophomonas maltophilia, caused by ready-to-use commercial syringes containing liquid lithium and heparin for arterial blood gas collection in a university hospital.MethodsUpon detecting an increase in Stenotrophomonas maltophilia growth in blood cultures between 15.09.2021 and 19.11.2021, an outbreak analysis and a case-control study (52 patients for the case group, 56 patients for the control group) were performed considering risk factors for bacteremia. Samples from possible foci for bacteremia were also cultured. Growing bacteria were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The genetic linkage and clonal relationship isolates were investigated with pulsed-field gel electrophoresis (PFGE) in the reference laboratory.ResultsIn the case-control study, the odds ratio for the central venous catheter [3.38 (95% confidence interval [CI]: 1.444, 8.705 ; p = 0.006)], for surgery [3.387 (95% confidence interval [CI]: 1.370, 8.373 ; p = 0.008)] and for arterial blood gas collection history [18.584 (95% confidence interval [CI]:4.086, 84.197; p < 0.001)] were identified as significant risk factors. Stenotrophomonas maltophilia growth was found in ready-to-use commercial syringes used for arterial blood gas collection. Molecular analysis showed that the growths in the samples taken from commercial syringes and the growths from blood cultures were the same. It was decided that the epidemic occurred because the method for sterilization of heparinized liquid preparations were not suitable. After discontinuing the use of the kits with this lot number, the outbreak was brought under control.ConclusionsAccording to our results, disposable or sterile medical equipment should be included as a risk factor in outbreak analyses. The method by which injectors containing liquids, such as heparin, are sterilized should be reviewed. Our study also revealed the importance of the cooperation of the infection control team with the microbiology laboratory.

Preparation and Characterization of H-Shaped Polylactide.

An H-polymer has an architecture that consists of four branches symmetrically attached to the ends of a polymer backbone, similar in shape to the letter "H". Here, a renewable H-polymer efficiently synthesized using only ring-opening transesterification is demonstrated. The strategy relies on a tetrafunctional poly(±-lactide) macroinitiator, from which four poly(±-lactide) branches are grown simultaneously. 1H NMR spectroscopy, size exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization (MALDI) spectrometry were used to verify the macroinitiator purity. Branch growth was probed using 1H NMR spectroscopy and SEC to reveal unique transesterification phenomena that can be controlled to yield architecturally pure or more complex materials. H-shaped PLA was prepared at the multigram scale with a weight-average molar mass Mw > 100 kg/mol and low dispersity Đ < 1.15. Purification involved routine precipitations steps, which yielded products that were architecturally relatively pure (∼93%). Small-amplitude oscillatory shear and extensional rheology measurements demonstrate the unique viscoelastic behavior associated with the H-shaped architecture.

In vitro cholinesterases, tyrosinase, α-glucosidase inhibitory and anticancer effects of novel silicon phthalocyanines containing geraniol/phytol

The axially silicon phthalocyanines, consisting of different acyclic hydrocarbons, were generated by SiPcCl2 reacting with either geraniol or phytol in toluene in the presence of NaH. FT-IR, UV–Vis, MALDI TOF, and NMR spectroscopy all provided information about the structures of silicon phthalocyanines. The cholinesterase, tyrosinase, and α-glucosidase inhibitory effects of G-SiPc and P-SiPc were tested using spectrophotometric assays. The IC50 values of G-SiPc and P-SiPc were 7.22 ± 0.53 and 11.28 ± 2.73 µM for AChE; 70.05 ± 3.00 and 60.55 ± 3.80 µM for BuChE. In addition, G-SiPc and P-SiPc inhibited the tyrosinase enzyme with IC50 values of 280.36 ± 7.92 µM and 40.51 ± 3.88 µM, respectively. P-SiPc had about seven times stronger inhibitory properties than G-SiPc. The substances exhibited less inhibition than acarbose in the α-glucosidase inhibitory assay. Finally, the cytotoxic effects of G-SiPc and P-SiPc were investigated using the MTT assay on L-929 mouse fibroblast cells as normal cells. G-SiPc and P-SiPc did not reach IC50 values up to 100 μM.

Antibody-Drug Conjugate Made of Zoledronic Acid and the Anti-CD30 Brentuximab-Vedotin Exert Anti-Lymphoma and Immunostimulating Effects.

Relevant advances have been made in the management of relapsed/refractory (r/r) Hodgkin Lymphomas (HL) with the use of the anti-CD30 antibody-drug conjugate (ADC) brentuximab-vedotin (Bre-Ved). Unfortunately, most patients eventually progress despite the excellent response rates and tolerability. In this report, we describe an ADC composed of the aminobisphosphonate zoledronic acid (ZA) conjugated to Bre-Ved by binding the free amino groups of this antibody with the phosphoric group of ZA. Liquid chromatography-mass spectrometry, inductively coupled plasma-mass spectrometry, and matrix-assisted laser desorption ionization-mass spectrometry analyses confirmed the covalent linkage between the antibody and ZA. The novel ADC has been tested for its reactivity with the HL/CD30+ lymphoblastoid cell lines (KMH2, L428, L540, HS445, and RPMI6666), showing a better titration than native Bre-Ved. Once the HL-cells are entered, the ADC co-localizes with the lysosomal LAMP1 in the intracellular vesicles. Also, this ADC exerted a stronger anti-proliferative and pro-apoptotic (about one log fold) effect on HL-cell proliferation compared to the native antibody Bre-Ved. Eventually, Bre-Ved-ZA ADC, in contrast with the native antibody, can trigger the proliferation and activation of cytolytic activity of effector-memory Vδ2 T-lymphocytes against HL-cell lines. These findings may support the potential use of this ADC in the management of r/r HL.

High Diversity of Curtobacterium flaccumfaciens in Poinsettia and Detection of Three Pathogenicity Plasmids for Identification of C. flaccumfaciens pv. poinsettiae

Outbreaks in Europe have raised concerns about poinsettia bacterial canker caused by Curtobacterium flaccumfaciens pv. poinsettiae, described in the United States. Using a semiselective medium containing aztreonam and fosfomycin and selection during isolation based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra, 424 Curtobacterium strains were isolated from Belgian poinsettias of African and European origin. Different populations coexisted: 130 strains were identified as C. flaccumfaciens with scores ≥2.0 and 294 with scores &lt;2.0 or as another Curtobacterium. MALDI-TOF MS libraries constructed using similar medium and extraction procedure and a pathogenicity test on poinsettia were used to screen collections from poinsettia and wheat, a possible alternative host, for C. flaccumfaciens pv. poinsettiae. The concatenated recA- gyrB partial sequences showed that 114 poinsettia or wheat strains belonged to different Curtobacterium species. Eighty-eight nonpathogenic strains and four U.S. strains from litter were intermingled with bean pathogenic C. flaccumfaciens pv. flaccumfaciens strains in the three genetic groups of C. flaccumfaciens but lacked their pathogenicity markers. Four new European recA- gyrB sequatypes of C. flaccumfaciens pv. poinsettiae, obtained from symptomatic and asymptomatic poinsettias, were distinguished from three American sequatypes. Six sequatypes were pathogenic in tests, belonged to genetic groups related to two different genomospecies, and possessed a plasmid. Sequencing of six plasmids revealed three related plasmids containing proteases and a polygalacturonase found only in strains pathogenic in pathogenicity tests and specifically identified by polygalacturonase-based PCR and loop-mediated isothermal amplification assays. Similarities between plant and litter Curtobacterium and the role of plasmids in pathogenicity and punctual transmission of pathogenicity among heterogeneous C. flaccumfaciens are suggested.

First report of azole-resistant Aspergillus species in poultry feed in Kabale, South West, Uganda

Widespread use of azoles in clinical and environmental settings has favored the selection of azole-resistant Aspergillus species necessitating antifungal susceptibility studies understanding their prevalence and resistance profiles in those settings. However, there is still limited information available on these strains in Africa. The objective of this study was to assess the environmental prevalence of Azole-resistance Aspergillus strains in poultry feed in Kabale, South West Uganda. The study evaluated the susceptibility profile of Aspergillus and other fungi isolates from 10 poultry feeds across 10 different commercial outlets. Isolates were first identified using rDNA 18S genomic sequencing via ITS 1 and 2 primer combination and confirmed using Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). Seventeen fungal strains were identified of which Aspergillus flavus was the most prevalent. All Aspergillus and one Penicillium isolates were further screened for azole-resistance using azole-containing agar plates (itraconazole, voriconazole, posaconazole, isavuconazole) and fluconazole antifungal susceptibility strip method at 4 mg/L, which was the first line of treatment. Aspergillus chevalieri, A. tamari, A. flavus, P. citrinum fungi isolates showed resistance to fluconazole. The EUCAST susceptibility testing method did not show any of the isolates resistant to tested azoles (itraconazole, voriconazole, posaconazole, isavoriconazole) or amphotericin B. Overall, we found resistance to fluconazole. This study is the first investigative study of Aspergillus species in poultry feed in Kabale, South West, Uganda.

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Delay in the diagnosis of Brucella abortus bacteremia in a nonendemic country: a case report

BackgroundIt is challenging to diagnose brucellosis in nonendemic regions because it is a nonspecific febrile disease. The accurate identification of Brucella spp. in clinical microbiology laboratories (CMLs) continues to pose difficulties. Most reports of misidentification are for B. melitensis, and we report a rare case of misidentified B. abortus.Case presentationA 67-year-old man visited an outpatient clinic complaining of fatigue, fever, and weight loss. The patient had a history of slaughtering cows with brucellosis one year prior, and his Brucella antibody tests were negative twice. After blood culture, the administration of doxycycline and rifampin was initiated. The patient was hospitalized due to a positive blood culture. Gram-negative coccobacilli were detected in aerobic blood culture bottles, but the CML's lack of experience with Brucella prevented appropriate further testing. Inaccurate identification results were obtained for a GN ID card of VITEK 2 (bioMérieux, USA) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) using a MALDI Biotyper (Bruker, Germany). The strain showed 100.0% identity with Brucella spp. according to 16S rRNA sequencing. MALDI–TOF MS peaks were reanalyzed using the CDC MicrobeNet database to determine Brucella spp. (score value: 2.023). The patient was discharged after nine days of hospitalization and improved after maintaining only doxycycline for six weeks. The isolate was also identified as Brucella abortus by genomic evidence.ConclusionAutomated identification instruments and MALDI–TOF MS are widely used to identify bacteria in CMLs, but there are limitations in accurately identifying Brucella spp. It is important for CMLs to be aware of the possibility of brucellosis through communication with clinicians. Performing an analysis with an additional well-curated MALDI–TOF MS database such as Bruker security-relevant (SR) database or CDC MicrobeNet database is helpful for quickly identifying the genus Brucella.

DNA Walker-Driven Mass Nanotag Assembly System for Simultaneously Profiling Dual Markers of Oxidative Stress at Different Cellular Locations.

Simultaneous profiling of redox-regulated markers at different cellular sublocations is of great significance for unraveling the upstream and downstream molecular mechanisms of oxidative stress in living cells. Herein, by synchronizing dual target-triggered DNA machineries in one nanoentity, we engineered a DNA walker-driven mass nanotag (MNT) assembly system (w-MNT-AS) that can be sequentially activated by oxidative stress-associated mucin 1 (MUC1) and apurinic/apyrimidinic endonuclease 1 (APE1) from plasma membrane to cytoplasm and induce recycled assembly of MNTs for multiplex detection of the two markers by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In the working cascade, the sensing process governs the separate activation of w-MNT-AS by MUC1 and APE1 in diverse locations, while the assembly process contributes to the parallel amplification of the ion signal of the characteristic mass tags. In this manner, the differences between MCF-7, HeLa, HepG2, and L02 cells in membrane MUC1 expression and cytoplasmic APE1 activation were fully characterized. Furthermore, the oxidative stress level and dynamics caused by exogenous H2O2, doxorubicin, and simvastatin were comprehensively demonstrated by tracking the fate of the two markers across different cellular locations. The proposed w-MNT-AS coupled MS method provides an effective route to probe multiple functional molecules that lie at different locations while participating in the same cellular event, facilitating the mechanistic studies on cellular response to oxidative stress and other disease-related cellular processes.

Biological Activity and Phytochemical Characteristics of Star Anise (Illicium verum) Essential Oil and Its Anti-Salmonella Activity on Sous Vide Pumpkin Model.

Illicium verum, commonly known as star anise, represents one of the notable botanical species and is recognized for its rich reservoir of diverse bioactive compounds. Beyond its culinary application as a spice, this plant has been extensively utilized in traditional medicine. Given the contemporary emphasis on incorporating natural resources into food production, particularly essential oils, to enhance sensory attributes and extend shelf life, our study seeks to elucidate the chemical composition and evaluate the antibacterial (in vitro, in situ) and insecticidal properties of Illicium verum essential oil (IVEO). Also, microbiological analyses of pumpkin sous vide treated with IVEO after inoculation of Salmonella enterica were evaluated after 1 and 7 days of study. GC/MS analysis revealed a significantly high amount of (E)-anethole (88.4%) in the investigated EO. The disc diffusion method shows that the antibacterial activity of the IVEO ranged from 5.33 (Streptococcus constellatus) to 10.33 mm (Citrobacter freundii). The lowest minimal inhibition concentration was found against E. coli and the minimum biofilm inhibition concertation was found against S. enterica. In the vapor phase, the best antimicrobial activity was found against E. coli in the pears model and against S. sonei in the beetroot model. The application of the sous vide method in combination with IVEO application decreased the number of microbial counts and eliminated the growth of S. enterica. The most isolated microbiota identified from the sous vide pumpkin were Bacillus amyloliquefaciens, B. cereus, B. licheniformis, and Ralstonia picketii. Modifications to the protein composition of biofilm-forming bacteria S. enterica were suggested by the MALDI TOF MS instigations. The IVEO showed insecticidal potential against Harmonia axyridis. Thanks to the properties of IVEO, our results suggest it can be used in the food industry as a natural supplement to extend the shelf life of foods and as a natural insecticide.