MALDI Research Articles

FSpatial metabolomics to discover hypertrophic scar relevant metabolic alterations and potential therapeutic strategies: A preliminary study

Spatially mapping the metabolic remodeling of hypertrophic scar and surrounding normal skin tissues has the potential to enhance our comprehension of scar formation and aid in the advancement of therapeutic interventions. In this study, we employed matrix-assisted laser desorption/ionization (MALDI), a mass spectrometry imaging technique, to visualize the hierarchical distribution of metabolites within sections of hypertrophic scar and surrounding normal skin tissues. A comprehensive analysis identified a total of 1631 metabolites in these tissues. The top four classes that were identified included benzene and substituted derivatives, heterocyclic compounds, amino acids and its metabolites, and glycerophospholipids. In hypertrophic scar tissues, 22 metabolites were upregulated and 66 metabolites were downregulated. MetaboAnalyst pathway analysis indicated that glycerophospholipid metabolism was primarily associated with these altered 88 metabolites. Subsequently, six metabolites were selected, their spatial characteristics were analyzed, and they were individually added to the cell culture medium of primary hypertrophic scar fibroblasts. The preliminary findings of this study demonstrate that specific concentrations of 1-pyrrolidinecarboxamide, 2-benzylideneheptanal, glycerol trioleate, Lyso-PAF C-16, and moxonidine effectively inhibited the expressions of COL1A1, COL1A2, COL3A1, and ACTA2. These bioactive metabolites exhibit mild and non-toxic properties, along with favorable pharmacokinetics and pharmacodynamics, making them promising candidates for drug development. Consequently, this research offers novel therapeutic insights for hypertrophic scar treatment.

Polycondensation, Cyclization and Disproportionation of Solid Poly(L-lactide) Trifluoroethyl Esters and the Simultaneous Formation of Extended Chain Crystals and Extended Ring Crystals

Two poly(L-lactide)s (PLAs) with a degree of polymerization (DP) of 20 or 100 were prepared by trifluoroethanol-initiated ring-opening polymerization (ROP) catalyzed by tin(II) 2-ethyl hexanoate (SnOct2). These PLAs were annealed at 140 °C or at 160 °C in the presence of SnOct2, and the changes in topology and molecular weight distribution (MWD) were monitored by matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry and gel permeation chromatography (GPC). For the PLA with a DP 20, the main reaction was polycondensation combined with higher dispersities. In the case of the DP 100, PLA polycondensation was combined with disproportionation and the formation of a new MWD maximum around m/z 3 500. In addition, extensive cyclization occurred, and the resulting cyclic PLAs crystallized separately from the linear chains in the form of extended ring crystals. These results also suggest that both extended chain and extended ring crystals posses the same crystal thickness as a result of thermodynamically controlled transesterification in the solid state.

A rare case of cerebellar abscess caused by Nocardia cyriacigeorgica

Background: Gram-positive opportunistic bacteria of the Nocardia species are responsible for a large spectrum of infections, such as pneumonia, skin infections, and more widespread conditions, including brain abscesses. Case Description: A 67-year-old male patient suffered from headache, gait disorder, and vertigo for a week before admission to our department. An enhanced magnetic resonance imaging scan revealed a mediosagittal hyperintense infratentorial lesion with concomitant compression of the fourth ventricle. The patient underwent surgical treatment with general anesthesia. The frozen section did not reveal any tumoral tissue but rather a purulent content. He was comatose on the 1st postoperative day, and he underwent a follow-up computed tomography (CT) scan, which revealed triventricular hydrocephalus. The external ventricular drain was performed, and a follow-up CT scan revealed significant improvement of hydrocephalus. Matrix-Assisted Laser Desorption Ionization Time of Flight did not reveal any causative agent from the intraoperative content, but the 16s ribosomal DNA method confirmed Nocardia cyriacigeorgica. The patient was intravenously treated with ceftriaxone and trimethoprim/sulfamethoxazole and died on the 5th postoperative day. Conclusion: Nocardiosis presents a rare Gram-positive bacterial infection that typically affects immunocompromised hosts. Nocardia-caused brain abscesses present a significant challenge in its treatment for its atypical presentation and slow culture growth.

Quantitative sampling by IR-MALDESI is not susceptible to tissue heterogeneity in a multi-organ model.

Quantitative mass spectrometry imaging (qMSI) provides the relative or absolute analyte quantities in a biological specimen in a spatially resolved manner. However, the chemical complexity and physical structure of biological specimens often require one to precisely account for matrix effects in qMSI platforms. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) completely ablates a volume of cryosectioned tissue. This enables the use of a normalization standard that is sprayed underneath the tissue for qMSI applications. Complete sampling has shown to be a significant advantage for qMSI by IR-MALDESI; however, the impact of high tissue heterogeneity has not been systematically studied or quantified. The bias introduced by tissue heterogeneity was investigated by uniformly spraying standards beneath and on top of a whole-body zebrafish section. The quantitative relationship between the signals of the two standards was investigated across this multi-organ model to serve future qMSI experiments by IR-MALDESI and other laser ablation-based sampling methods. The overall ratio between the standards sprayed on top of and beneath the tissue sections remained constant across the entire whole-body section despite significant tissue heterogeneity (e.g., gills, heart, and liver). Additionally, we noted that thinner and/or sucrose-embedded tissues improved these ratios, which will inform future qMSI investigations.

Biodegradable Polyurethane Foams Based on Polyols Obtained from Cellulose and Its Hydroxypropyl Derivative

Three methods of cellulose-derived polyol synthesis were elaborated. The suitable substrates were (hydroxypropyl)cellulose or cellulose, which were hydroxyalkylated in reactions with glycidol and ethylene carbonate in triethylene glycol or in water. The products were characterized by IR, 1H NMR, and MALDI ToF spectroscopies. For all polyols, IR spectra showed strong bands at 1060 cm−1 from the ether group formed upon the ring opening of GL and EC. The polyol obtained from (hydroxypropyl)cellulose in the triethylene glycol solvent was accompanied by oligomeric products of glycol hydroxyalkylation and oligomeric glycidol. The polyol obtained by the hydroxyalkylation of cellulose with glycidol and ethylene carbonate in the water contained units of hydroxyalkylated cellulose and products of hydroxyalkylation of water. The physical properties of the obtained polyols, like density, viscosity, and surface tension, were determined. The polyols were then used to obtain rigid polyurethane foams. The foams have apparent density, water uptake, and polymerization shrinkage similar to classic rigid PUFs. The foams showed advantageous thermal resistance in comparison with classic ones. After thermal exposure, their compressive strength improved. The biodegradation of the obtained materials was tested by a respirometric method in standard soil conditions by the measurement of biological oxygen demand and also using the cellulases or the enzymes responsible for cellulose degradation. It has been found that polyols are totally biodegradable within one month of exposure, while the foams obtained thereof are at least 50% biodegraded in the same conditions. The enzymatic biodegradation of the PUFs by the action of microbial cellulase was confirmed.

Membrane-based preparation for mass spectrometry imaging of cultures of bacteria.

The study of the dialogue between microorganisms at the molecular level is becoming essential to understand their relationship (antagonist, neutral, or beneficial interactions) and its impact on the organization of the microbial community. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization (MALDI) is a technique that reveals the spatial distribution of molecules on a sample surface that may be involved in interactions between organisms. An experimental limitation to perform MALDI MSI is a flat sample surface, which in many cases could not be achieved for bacterial colonies such as filamentous bacteria (e.g., Streptomyces). In addition, sample heterogeneity affects sample dryness and MALDI matrix deposition prior to MSI. To avoid such problems, we introduce an additional step in the sample preparation. A polymeric membrane is interposed between the microorganisms and the agar-based culture medium, allowing the removal of bacterial colonies prior to MSI of the homogeneous culture medium. A proof of concept was evaluated on Streptomyces ambofaciens (a soil bacterium) cultures on solid media. As the mycelium was removed at the same time as the polymeric membrane, the metabolites released into the medium were spatially resolved by MALDI MSI. In addition, extraction of the recovered mycelium from the membrane confirmed the identification of the metabolites by ESI MS/MS analysis. This approach allows both the spatial distribution of metabolites produced by microorganisms in an agar medium to be studied under well-controlled sample preparation and their structure to be elucidated. This capability is illustrated using desferrioxamine E, a siderophore produced by S. ambofaciens.

Development of a MALDI-TOF MS model for differentiating haemorrhagic septicaemia-causing strains of Pasteurella multocida from other capsular groups

Pasteurella multocida capsular types A, D, and F cause disease in many animal hosts, including bovine respiratory disease in cattle, which is one of the most globally significant animal diseases. Additionally, P. multocida capsular types B and E cause haemorrhagic septicaemia, a devastating disease primarily of cattle, water buffalo, and bison that develops rapidly with high mortality. Haemorrhagic septicaemia mostly occurs in developing countries and has potential to emerge elsewhere in the world. The diagnosis of haemorrhagic septicaemia currently requires recognition of compatible gross or histologic lesions and serotyping or molecular characterization of strains. In this study, we performed genomic characterization of 84 P. multocida strains, which were then used to develop and validate a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) biomarker-based method for differentiating non-haemorrhagic septicaemia strains of P. multocida from haemorrhagic septicaemia-causing strains. Haemorrhagic septicaemia strain types B:2,5, E:2,5, and B:3,4 were used to maximize diversity. Three automated classification models were generated and then used to develop an assisted model, which utilized two peaks (6419 and 7729 m/z) to accurately differentiate non-haemorrhagic septicaemia-causing strains from haemorrhagic septicaemia-causing strains of P. multocida. The assisted model performed with 98.2 % accuracy for non-haemorrhagic septicaemia strains, 100 % accuracy for classic B:2,5 and E:2,5 strains, and 84.4 % accuracy for combined haemorrhagic septicaemia-causing strains (B:2,5, E:2,5, and B:3,4) with an overall accuracy of 96.9 %. Our results suggest that MALDI-TOF MS may be used to routinely screen P. multocida isolated from diagnostic cases for initial identification of haemorrhagic septicaemia-causing strains, and to determine whether additional characterizations are warranted.

Integration of MALDI glycotyping and NMR analysis to uncover an O-antigen substructure from pathogenic Escherichia coli O111

Escherichia coli O111 serotype is a critical pathogenic E. coli strain that causes severe, potentially fatal complications. Despite its reported variation, only one structure of the O-antigen polysaccharide from E. coli O111 has been reported. Here, a substructure of the O-antigen from E. coli O111 was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and NMR analysis. MALDI glycotyping revealed differing O-antigen repeating unit masses of Δm/z 787 and 828 in the E. coli strains and lipopolysaccharides from the O111 serogroup. This variation was caused by the replacement of the hexose residue with hexosamine in the repeating units, which was further confirmed by LIFT-TOF/TOF analysis. Structural elucidation of the O111 substructure by NMR analysis further demonstrated replacement of the hydroxyl group with an N-acetyl group on the terminal glucose residue of the O-antigen pentasaccharide repeating unit. To our knowledge, this study is the first to provide a detailed structural analysis of a new O-antigen substructure from the E. coli O111 serogroup.

Use of a hydrosilylation reaction for the preparation of structure-controlled boroxine-based polyborosiloxanes

The structure of organosilicon vinyl-containing boroxine was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Its thermal properties were studied using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) methods. The ability of boroxine to undergo hydrosilylation using the Karstedt's catalyst was investigated on various organosilicon substrates. Boroxine was shown to be involved in these reactions as a cross-linking agent. The formation of dynamic boroxine cross-links allowed the analysis of the reaction products by NMR spectroscopy, including the use of polyfunctional hydride-containing reagents. In all cases the addition proceeds selectively to the β-position and with complete conversion. The structure of the resulting polyborosiloxane containing 1.4 mol% of modified units was also confirmed by 1H NMR spectroscopy, and its thermal and rheological properties were studied.

Poxylated stereocomplexes from PEtOx-b-PLA diblock copolymers

An ω-hydroxyl terminated poly(2-ethyl-2-oxazoline) (PEtOx) was obtained by termination of the cationic ring-opening polymerization of 2-ethyl-2-oxazoline with acetate and subsequent transesterification with methanol. The resulting PEtOx-OH featuring a degree of polymerization (DP) of 20 was used as a macroinitiator for the ring-opening polymerization of l-lactide as well as d-lactide. The resulting PEtOx-b-PLA block copolymers featured DP values of 25, 50, 100 and 150, respectively, and were characterized by means of size exclusion chromatography, 1H NMR spectroscopy as well as matrix-assisted laser desorption ionization mass spectrometry. Differential scanning calorimetry, wide-angle x-ray scattering and polarized light microscopy indicated the formation of stereocomplexes in racemic blends of PEtOx-b-PLA with opposing chirality. Thereby, the amorphous PEtOx block did not affect the modification of the stereocomplex crystallites, as shown by comparison with data obtained from PLA homopolymer stereocomplexes. Similar to those, the degree of crystallinity and melting temperature decreased with increasing molar mass of the PLA in the stereocomplexes formed from PEtOx-b-PLA block copolymers.

Enhanced detection of Pythium insidiosum via lipid profiling with matrix-assisted laser desorption ionization time of flight mass spectrometry

Pythiosis is a severe disease in humans and animals globally, caused by the pathogenic oomycete Pythium insidiosum. Early and accurate detection is crucial for effective treatment, but traditional diagnostic methods have limitations. This study presents an alternative approach using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) for lipid profiling to efficiently identify P. insidiosum. The study involved extracting microbial lipid components using optimized chloroform: methanol biphasic method and creating a lipid profile database with samples from 30 P. insidiosum isolates and 50 various fungi. The methodology was validated on 25 blinded samples for assay detection performance. Unique lipid profiles allowed species-specific identification with high efficiency: scores ≥ 2.682 indicated P. insidiosum, scores ≤ 2.512 suggested fungi, and scores in between pointed to other oomycetes. The assay demonstrated sensitivity, specificity, and accuracy of 100%, 80%, and 88%, respectively, for detecting P. insidiosum. The limited detection specificity was due to false positive samples from closely related Pythium species, which are not a significant clinical concern. The findings show that MALDI-TOF MS lipid profiling can efficiently identify P. insidiosum, offering significant advantages in sample preparation, stability, and reproducibility over protein profile-based methods. This study marks the first instance of lipid profiles being reported for P. insidiosum, paving the way for clinical use in improving accurate detection and facilitating timely treatment interventions.

Evaluation of chelating agents based on pyridine-azacrown compounds H4PATA, PATAM, and H4PATPA for 68Ga and 177Lu

In this article, we present the synthesis and characterization of three macrocyclic chelators, H4PATA, PATAM, and H4PATPA, based on a pyridine-azacrown compound. Their complexation with 68Ga and 177Lu has been thoroughly investigated using MALDI TOF MS, 1H NMR spectroscopy, radiolabeling studies, and experiments in vitro with fetal bovine serum and a 1000-fold molar excess of H4EDTA. Our studies have shown that the chelators H4PATA and H4PATPA form complexes at room temperature with both radionuclides (RCY >80 % and >90 % for complexes with H4PATA and H4PATPA after 30 min, respectively). The chelator PATAM requires high temperatures (95 °C) for complexation. In vitro stability assays in fetal bovine serum as well as H4EDTA-challenge revealed that transchelation occurs for all complexes with 68Ga. However, complexes of the ligands H4PATA and PATAM with 177Lu were found stable. Thus, taking into account the radiolabeling at room temperature and in vitro stability of the complex [177Lu]Lu·PATA, our investigations revealed the chelator H4PATA is a candidate for radiopharmaceutical use with 177Lu.

Bartonella Species in Small Mammals in Turkey: Bartonella bilalgolemii sp. nov. Isolated from a Ural Field Mouse (Apodemus uralensis).

Background: The genus Bartonella is composed of Gram-negative, fastidious, facultative intracellular bacteria that can cause bacteremia in mammals and various disorders in humans. Rodents have been reported as reservoirs of more than 30 Bartonella species, seven of which cause zoonotic infections. Materials and Methods: In the present study, the isolation of Bartonella sp. was attempted from 150 spleen samples from 13 rodent species (mostly Apodemus species) from three geographically different regions in Turkey. Results: Bartonella sp. was successfully isolated from 65 of these 150 samples (43%). The prevalences of Bartonella sp. in tested rodents in the regions of Giresun, Yozgat, and Burdur were 68%, 44%, and 16%, respectively. Using polymerase chain reaction/sequence analysis of the citrate synthase-coding gene (gltA), Bartonellaisolates were classified seven species including B. taylorii, B. grahamii, B. birtlesii, B. mastomydis, and three putatively new Bartonella species. We performed further identification techniques for one of the three Bartonella species that were different from the validated Bartonella species according to the gltA sequence analysis. Conclusion: Here, we report the genomic and phenotypic characterization of Bartonella sp. strain G70 that was isolated from the splenic tissue of an Apodemus uralensis (Pallas 1881), the Ural field mouse, captured in the Giresun region of northeastern Turkey. Bartonella sp. strainG70 (RSKK 22001) was characterized by whole genome and partial gene (gltA, 16S ribosomal RNA) sequencing and comparison, scanning electron microscopy, biochemical tests, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This novel Bartonella is a Gram-negative, rod-shaped bacterium and has neither flagella nor pili. The genome from strain G70 was 1,606,969-bp-long with a G + C content of 35.7%. Bartonella rochalimae was found to be the closest phylogenetic relative of strain G70 (OrthoANI = 90.5%, digital DNA-DNA hybridization = 41.4%). We therefore propose that this new species be named Bartonella bilalgolemii sp. nov. with strain G70T as the type strain.

Use of stable isotope combined with intact cell lipidomic by routine MALDI mass spectrometry analysis for rapid drug susceptibility assay in mycobacteria.

Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile. Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc2155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M.smegmatis mc2155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (D2O) and either 1 or 2 times the minimal inhibitory concentration (MIC50) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile. Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5h, a process that would take 72 h using the conventional broth microdilution method. We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining D2O stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.

Evaluation of the Activity of Triazoles Against Non-fumigatus Aspergillus and Cryptic Aspergillus Species Causing Invasive Infections Tested in the SENTRY Program.

The activity of isavuconazole and other triazoles against non-fumigatus (non-AFM) Aspergillus causing invasive aspergillosis was evaluated. A total of 390 non-AFM isolates were collected (1/patient) in 2017-2021 from 41 hospitals. Isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and/or internal spacer region/β-tubulin sequencing and tested by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. CLSI epidemiological cutoff values were applied, where available. Isavuconazole showed activity against Aspergillus sections Flavi (n = 122; minimum inhibitory concentration [MIC]50/90, 0.5/1 mg/L), Terrei (n = 57; MIC50/90, 0.5/0.5 mg/L), Nidulantes (n = 34; MIC50/90, 0.12/0.25 mg/L), Versicolores (n = 7; MIC50, 1 mg/L), and Circumdati (n = 2; MIC range, 0.12-2 mg/L). Similar activity was displayed by other triazoles against those Aspergillus sections. Most of the isolates from Aspergillus sections Fumigati (n = 9), Nigri (n = 146), and Usti (n = 12) exhibited elevated MIC values to isavuconazole (MIC50/90, 2/-, 2/4, and 2/8 mg/L), voriconazole (MIC50/90, 2/-, 1/2, and 4/8 mg/L), itraconazole (MIC50/90, 2/-, 2/4, and 8/>8 mg/L), and posaconazole (MIC50/90, 0.5/-, 0.5/1, and >8/>8 mg/L), respectively. Isavuconazole was active (MIC values, ≤1 mg/L) against Aspergillus parasiticus, Aspergillus tamarii, Aspergillus nomius, Aspergillus nidulans, Aspergillus unguis, Aspergillus terreus, Aspergillus alabamensis, and Aspergillus hortai, while isavuconazole MIC values between 2 and 8 mg/L were observed against cryptic isolates from Aspergillus section Fumigati. Isavuconazole inhibited 96.1% of Aspergillus niger and 80.0% of Aspergillus tubingensis at ≤4 mg/L, the CLSI wild-type cutoff value for A niger. Voriconazole, itraconazole, and posaconazole showed similar activity to isavuconazole against most cryptic species. Isavuconazole exhibited potent in vitro activity against non-AFM; however, the activity of triazoles varies among and within cryptic species.

Severe co-infection caused by difficult-to-diagnose hypermucoviscous Klebsiella pneumoniae K1-ST82 in a patient with COVID-19: a case report

BackgroundCo-infection with Klebsiella pneumoniae presents a significant concern in hospitalized patients with coronavirus disease (COVID-19), increasing the risk of severe disease progression. Hypervirulent (hv) and hypermucoviscous (hm) K. pneumoniae (Kp) has gained prominence in Asia due to its capacity to cause invasive community-acquired infections. However, recognition of hvKp/hmKp co-infections in the context of COVID-19 remains limited. We report a severe case of rapidly progressing co-infection with hmKp exhibiting “difficult-to-diagnose” phenotypes in a hospitalized patient with COVID-19.Case presentationA 61-year-old woman with COVID-19 initially exhibited mild symptoms resembling the common cold. However, her condition rapidly deteriorated over 7 days, leading to hospital admission with the development of dyspnea. The patient required supplemental oxygen, antibiotic treatment, and mechanical ventilation. Gram-negative bacteria with atypical phenotypes were isolated from alveolar lavage fluid and blood cultures. Both strains formed small, glossy, non-lactose-fermenting colonies on clinically relevant media and were susceptible to ampicillin. Conventional biochemical tests failed to identify the Enterobacteriales strains owing to the urease-negative phenotype. Consequently, the identification of K. pneumoniae was difficult until matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed. A positive string test indicated mucoviscosity, but with variability in the material used for stretching colonies. Whole-genome sequencing performed on the MiSeq and GridION platforms revealed the blood-derived strain JARB-RN-0063 as belonging to serotype K1 and sequence type (ST) 82. The hvKp-associated genes rmpA and iroCD were located on a 5.0-Mb chromosome, and iucABCD-iutA was identified on a 217.9-kb IncFIB(K)/IncR-type plasmid. Therefore, JARB-RN-0063 was genetically classified as hvKp with a Kleborate virulence score of 3. The intrinsic penicillinase gene blaSHV was defective owing to an IS1F element insertion, resulting in the strain being atypically susceptible to ampicillin.ConclusionsThis is the first case of severe COVID-19-associated co-infection with a difficult-to-diagnose K. penummoniae strain. Notably, co-infection by the hmKp K1-ST82 clone exhibited atypical phenotypes, including stunted growth, non-lactose fermentation, urease-negative reaction, ampicillin susceptibility, and abnormal mucoviscosity, posing diagnostic challenges for clinical laboratories and impedes the identification of hvKp/hmKp. Delayed identification may worsen patient outcomes, highlighting the need for increased clinical awareness of such difficult-to-diagnose clones to prevent deterioration.

Accuracy of various matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based rapid identification methods—As a tool to augment diagnostic stewardship in blood culture laboratory, South India

BackgroundBlood stream infection is a medical emergency associated with high morbidity and mortality. Prompt identification of bloodstream infection-causing microorganisms directly from positive blood culture will significantly enhance patient care by reducing the turnaround time of pathogen recognition. MethodsA total of 256 freshly flagged positive blood culture bottles were subjected to Gram staining. Direct MALDI-TOF MS analysis was performed following sample preparation techniques such as lysis centrifugation, lysis filtration and VITEK® MS BC kit to directly identify microorganisms from positive blood cultures. Along with these short-term incubation methods of Choco spot and minute colony(8–10h) were also performed. All those positive bottles were identified by the routine (reference) laboratory method. Results177 isolates (69.14 %) were correctly identified by Lysis centrifugation, 163 isolates (63.67 %) were correctly identified by Lysis filtration, 206 isolates (80.47 %) were correctly identified by Choco spot,250 isolates (97.65 %) were correctly identified from minute colony (8–10h) of incubation. Of 162 isolates,115 isolates (70.99 %) were correctly identified by VITEK® MS Blood culture kit, (BioMérieux). VITEK® MS BC kit method revealed higher agreement with the kappa value of 0.697 than lysis centrifugation (0.672) followed by lysis filtration (0.611). ConclusionsIn house method of lysis centrifugation is found to be equivalent to VITEK® MS BC kit method and superior to lysis filtration method in correct direct identification of bacteria from positive blood cultures by MALDI-TOF MS analysis. As lysis centrifugation requires only 10 min of processing time as compared to overnight incubation, thus it offers a less expensive substitute for the VITEK® MS BC kit in the clinical laboratory. As a consequence of this study, we have implemented direct MALDI-TOF-based identification from positive BCs in our daily routine diagnostic management.

Genomic characterization of Pantoea dispersa A003 isolated from a clinical patient

IntroductionPantoea dispersa is a Gram-negative bacterium generally considered as a plant pathogen and rarely causes human infections. To date, there have been 13 studies that have documented clinical infections linked to P. dispersa, with a primary emphasis on the initial identification of this pathogen. The genomic features and the mechanisms underlying the pathogenesis of P. dispersa remain largely uninvestigated. In the present study, we describe a clinical infection caused by P. dispersa and provide the first genomic analysis of this bacterium.MethodsThe bacterial strain designated A003 was isolated from blood samples. Preliminary identification of strain A003 was conducted using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) technology (VITEK® MS, bioMérieux, France). Biochemical and antimicrobial susceptibility assessments were carried out utilizing the VITEK-2 compact automated microbial analysis system (bioMérieux, France). Additionally, whole genome sequencing and subsequent analysis were performed to further elucidate the potential pathogenicity.ResultsThe bacterial isolate was cultured overnight at 35°C in a CO2-enriched environment on Columbia blood agar, resulting in the appearance of a white, smooth, Gram-negative rod-shaped bacterium with diameters ranging from 1 to 2 mm. Identification of strain A003 was achieved with a high confidence level of 99.9% as P. dispersa using MALDI-TOF mass spectrometry. The average nucleotide identity (ANI) value between strain A003 and the reference strain P. dispersa DSM 30073T was 98.08%, while the DNA–DNA hybridization (DDH) value was 84.10% (Formula 2) based on genome sequencing data, which further confirmed that A003 belonged to the P. dispersa species. Comprehensive analysis revealed the presence of 372 virulence factors, 15 antibiotic resistance genes, 222 carbohydrate-active enzymes, and 666 genes related to Type III secretion system (T3SS) effector proteins, as identified through the core databases of VFDB (Virulence Factors of Pathogenic Bacteria), CARD (Comprehensive Antibiotic Resistance Database), CAZY (Carbohydrate-Active enZYmes Database), and T3SS. The virulence factors included type IV pili (TFP), type VI secretion system, flagella, iron uptake system, and ompA, which are implicated in bacterial pathogenicity. The antibiotic resistance profile indicated resistance to fluoroquinolones, cephalosporins, penams, macrolides, and aminoglycosides, as annotated by the CARD database.ConclusionThe draft genome sequenced represents the inaugural genomic sequence of P. dispersa derived from clinical sources. This advancement may enhance clinical practitioners’ comprehension of the organism’s clinical attributes and serve as a foundational resource for future research into its virulence, antibiotic resistance, and host–pathogen interactions.

Methodological aspects of correlative, multimodal, multiparametric breast cancer imaging: from data acquisition to image processing for AI-based radioproteomics in a preclinical setting

Preclinical high-field magnetic resonance imaging (MRI) systems offer a diverse array of MRI techniques, providing rich multiparametric MRI (mpMRI) platforms for studying numerous biological parameters. mpMRI platforms prove particularly indispensable when investigating tumors that exhibit profound intratumoral heterogeneity, such as breast cancer. A thoughtful comprehension of the origins of intratumoral heterogeneity is imperative for the judicious assessment of new targeted therapies and treatment interventions. Furthermore, when data from mpMRI are complemented with data from other in vivo imaging modalities, such as positron emission tomography (PET), and correlated with data from ex vivo modalities, such as matrix-assisted laser desorption imaging mass spectrometry (MALDI IMS), the in vivo parameters can be further elucidated at a molecular level and microscopic scale. Nevertheless, extracting meaningful scientific insights from such complex datasets necessitates the utilization of machine learning (ML) approaches to discern region-specific radiomic features. The development of correlative, multimodal imaging (CMI) workflows, such as one incorporating MRI, PET and MALDI IMS, is inherently challenging, given the many technological and methodological challenges related to multimodal data acquisition as well as the physiological limitations of the laboratory mice of the investigation. Standardization efforts in image acquisition and processing are required to increase the reproducibility and translatability of CMI data. To address the challenges of developing standardized CMI workflows and stimulate dialog regarding this area of need, we present a practical workflow to investigate tumor heterogeneity in breast cancer xenografts across various spatial scales. Our workflow entails simultaneous functional MRI and PET acquisitions in living mice, followed by correlation with post-imaging MALDI IMS and histologic data. Additionally, we propose data preprocessing steps for potential ML applications. We illustrate the feasibility of this workflow through two examples, showcasing its effectiveness in comparing in vivo and ex vivo images to evaluate tumor metabolism and hypoxia in mice with breast cancer xenografts.

Lack of Candida africana in Ugandan pregnant women: results from a pilot study using MALDI-ToF

BackgroundCandida africana is an emergent variant that has been listed as a new species or variety within the Candida albicans complex since 2001. It has a worldwide intra-albicans complex pooled prevalence of 1.67% and varies between 0 and 8% depending on geographical region. We present the results of a pilot study on its prevalence in Uganda.MethodologyWe conducted a cross-sectional study between March and June 2023. We recruited 4 pregnant women from Mulago Specialized Women and Neonatal Hospital, 102 from Kawempe National Referral Hospital, and 48 from Sebbi Hospital. Vaginal swabs were tested using microscopy, culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).ResultsThe prevalence of C. africana was zero. Out of the 103 isolates, the majority (81.553%) were identified as Candida albicans, followed by Nakeseomyces glabrata (13.592%) and Pichia kudriavzevii (1.942%). Cyberlindnera jadinii, Candida tropicalis, and Candida parapsilosis each accounted for 0.971% of the isolates.ConclusionThe prevalence of C. africana in Uganda is zero. However, large-scale cross-sectional studies, including studies involving the collection of vaginal samples from both urban and rural settings in Uganda and the use of both MALDI-TOF- and PCR-based laboratory methods, are needed to fully describe the public health burden of C. africana infections.