Assisted Laser Desorption Ionization Time Research Articles

Early organism identification by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) decreases the time to appropriate antibiotic modifications for common bacterial infections.

MALDI-TOF-MS facilitates the identification of microorganisms from positive cultures in a timely and accurate manner. It eliminates the necessity for the application of biochemicals and operates on the principle of proteomics. It decreases the time required to report culture results. Prompt detection and notification of the pathogen, prior to the disclosure of antimicrobial susceptibilities, could potentially shorten the duration until the initial antibiotic adjustment is necessary, thereby influencing patients' clinical prognoses. Fifty patients in the conventional arm and one hundred patients in the interventional arm were compared in a pre and post quasi-experimental study conducted at a tertiary care centre in North India. Patients with positive cultures from medical wards and intensive care units were included. Comparing the time to first antibiotic modification after culture positivity, MALDI-TOF-MS-based identification, and clinical outcomes in both arms was the primary objective. Antibiotic modifications, escalation, and de-escalation were all recorded. The intervention arm exhibited a substantially shorter median time to first antibiotic modification (2010 mins vs 2905 mins, p=0.002) than the conventional arm. In the interventional group, a total of 44 out of 100 antibiotic modifications were implemented. Of these, 19 (43.3%) were determined solely by the MALDI report, without the anticipation of susceptibility assessments. De-escalation of antibiotics constituted the pre-dominant form of modification (47.4%). The difference between the 27% and 32% mortality rates in the intervention arm and the conventional arm was not statistically significant (p=0.52). MALDI-TOF-MS facilitates the modification of antibiotics early on. The primary benefit lies in the reduction of superfluous antibiotic usage. Early organism identification and reporting prior to the availability of susceptibility results did not result in any mortality benefit. This strategy, when combined with a strong antimicrobial stewardship programme, can aid in the reduction of antibiotic use.

Hydrolytic Response of Beta-Amylase to Selected Starches when Adsorptively Immobilized on Agarose Gel

Fermentative microorganisms bring about transformative processes in food, thereby enhancing flavor and appearance indices, inhibiting undesirable components, improving shelf and storage life, as well as improving health and nutritional quality parameters. Traditional methods for identification of fermentative microorganisms involve cultivating and analyzing phenotypic characteristics, which are labor-intensive and time-consuming. Advanced molecular techniques such as polymerase chain reaction, whole gene sequencing, next generation sequencing, fluorescent in situ hybridization (FISH), Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), fourier transform infrared spectroscopy and hyper-spectral imaging offer rapid, cost-effective, and reliable identification of bacteria and fungi cultured on agar plates or in liquid media. Techniques, such as genotyping, examine the DNA sequence to determine the genetic makeup of microorganisms, alongside the grouping of isolates via application of phylogenetic tree similarity indices. Overall, modern molecular techniques have revolutionized the identification of fermentative microorganisms, providing efficient and more elaborate alternatives to traditional methods. In this review work, an attempt is made to examine the historical and analytical trends from the conventional methods to the most recent modern tools for effective identification of industrially-important fermentative microorganisms.

Recent Advances in the Identification and Characterization of Fermentative Microorganisms: An Exploratory Review

Fermentative microorganisms bring about transformative processes in food, thereby enhancing flavor and appearance indices, inhibiting undesirable components, improving shelf and storage life, as well as improving health and nutritional quality parameters. Traditional methods for identification of fermentative microorganisms involve cultivating and analyzing phenotypic characteristics, which are labor-intensive and time-consuming. Advanced molecular techniques such as polymerase chain reaction, whole gene sequencing, next generation sequencing, fluorescent in situ hybridization (FISH), Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), fourier transform infrared spectroscopy and hyper-spectral imaging offer rapid, cost-effective, and reliable identification of bacteria and fungi cultured on agar plates or in liquid media. Techniques, such as genotyping, examine the DNA sequence to determine the genetic makeup of microorganisms, alongside the grouping of isolates via application of phylogenetic tree similarity indices. Overall, modern molecular techniques have revolutionized the identification of fermentative microorganisms, providing efficient and more elaborate alternatives to traditional methods. In this review work, an attempt is made to examine the historical and analytical trends from the conventional methods to the most recent modern tools for effective identification of industrially-important fermentative microorganisms.

Red cabbage extract immobilized in bacterial cellulose film as an eco-friendly sensor to monitor microbial contamination and gamma irradiation of stored cucumbers

The aim of the present study is to develop a pH-sensing biopolymer film based on the immobilization of red cabbage extract (RCE) within bacterial cellulose (BC) to detect contamination and gamma radiation exposure in cucumbers. The results obtained show a sensitivity to pH changes for RCE in its aqueous form and that incorporated within BC films (RCE-BC), both showed color change correlated to bacterial growth (R2 = 0.91), this was supported with increase in pH values from 2 to 12 (R2 = 0.98). RCE and RCE-BC exposure to gamma radiation (0, 2.5, 5, 10, 15, 20, 25 kGy) resulted in gradual decrease in color that was more evident in RCE aqueous samples. To sense bacterial contamination of cucumbers, the total count was followed at 0, 5, 10 and 15 days in cold storage conditions and was found to reach 9.13 and 5.47 log cfu/mL for non-irradiated and 2 kGy irradiated samples, respectively. The main isolates detected throughout this storage period were identified as Pseudomonas fluorescens, Erwinia sp. Pantoea agglomerans using matrix assisted laser desorption ionization–time of flight-ms (MALDI–TOF–MS). Bacterial growth in stored irradiated cucumbers was detected by color change within 5 and 10 days of storage, after which there was no evident change. This is very useful since contamination within the early days of storage cannot be sensed with the naked eye. This study is the first to highlight utilizing RCE and RCE-BC as eco-friendly pH-sensing indicator films for intelligent food packaging to detect both food contamination and gamma preservation for refrigerator stored cucumbers.

Utilizing Direct MALDI-TOF MS from Positive Blood Culture for Diagnosing Anaerobic Bacteremia

Anaerobic bacteraemia is a significant cause of morbidity and mortality among hospitalized patients worldwide. Early identification of these microorganisms from blood culture is pivotal in diagnosing and can improve treatment and outcomes. We present a case of anaerobic bacteraemia in a patient with diabetic foot ulcer, which was detected rapidly by the Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) from a positive blood culture bottle in a laboratory setting with minimal facilities for anaerobic culture methods.

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.

Multidrug-Resistant Staphylococcus sp. and Enterococcus sp. in Municipal and Hospital Wastewater: A Longitudinal Study.

The objective of the study was to detect multidrug-resistant Staphylococcus sp. and Enterococcus sp. isolates in municipal and hospital wastewater and to determine their elimination or persistence after wastewater treatment. Between August 2021 and September 2022, raw and treated wastewater samples were collected at two hospital and two community wastewater treatment plants (WWTPs). In each season of the year, two treated and two raw wastewater samples were collected in duplicate at each of the WWTPs studied. Screening and presumptive identification of staphylococci and enterococci was performed using chromoagars, and identification was performed with the Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS®). Antimicrobial susceptibility was performed using VITEK 2® automated system. There were 56 wastewater samples obtained during the study period. A total of 182 Staphylococcus sp. and 248 Enterococcus sp. were identified. The highest frequency of Staphylococcus sp. isolation was in spring and summer (n = 129, 70.8%), and for Enterococcus sp. it was in autumn and winter (n = 143, 57.7%). Sixteen isolates of Staphylococcus sp. and sixty-three of Enterococcus sp. persisted during WWTP treatments. Thirteen species of staphylococci and seven species of enterococci were identified. Thirty-one isolates of Staphylococcus sp. and ninety-four of Enterococcus sp. were multidrug-resistant. Resistance to vancomycin (1.1%), linezolid (2.7%), and daptomycin (8.2%/10.9%%), and a lower susceptibility to tigecycline (2.7%), was observed. This study evidences the presence of Staphylococcus sp. and Enterococcus sp. resistant to antibiotics of last choice of clinical treatment, in community and hospital wastewater and their ability to survive WWTP treatment systems.

In vitro assessment of crude oil degradation by Acinetobacter junii and Alcanivorax xenomutans isolated from the coast of Ghana

This study was aimed at using in vitro microcosm experiments to assess crude oil degradation efficiency of Acinetobacter junii and Alcanivorax xenomutans isolated along Ghana's coast. Uncontaminated seawater from selected locations along the coast was used to isolate bacterial species by employing enrichment culture procedures with crude oil as the only carbon source. The isolates were identified by means of the extended direct colony transfer method of the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy (MALDI-TOF MS), as Acinetobacter junii, and Alcanivorax xenomutans. Remediation tests showed that Acinetobacter junii yielded degradation efficiencies of 27.59 %, 41.38 % and 57.47 %. Whereas efficiencies of 21.14 %, 32.18 % and 43.68 % were recorded by Alcanivorax xenomutans representing 15, 30 and 45 days respectively. Consortia of Acinetobacter junii, and Alcanivorax xenomutans also yielded 32.18 %, 48.28 % and 62.07 % for the selected days respectively. Phylogenetic characterization using ClustalW and BLAST of sequences generated from the Oxford Nanopore Sequencing technique, showed that the Ghanaian isolates clustered with Alcanivorax xenomutans and Acinetobacter junii species respectively. An analysis of the sequenced data for the 1394-bp portion of the 16S rRNA gene of the isolates revealed >99 % sequence identity with the isolates present on the GenBank database. The isolates of closest identity were Alcanivorax xenomutans and Acinetobacter junii with accession numbers, NR_133958.1 and KJ147060.1 respectively. Acinetobacter junii and Alcanivorax xenomutans isolated from Ghana's coast under pristine seawater conditions have therefore demonstrated their capacity to be used for the remediation of crude oil spills.

Carbapenemase and extended-spectrum beta-lactamase-producing bacteria in waters originating from a single landfill in Slovenia.

Groundwater, rainwater, and leachate associated with a single landfill were analysed to detect extended-spectrum beta-lactamase (ESBL)-producing and carbapenemase (CP)-producing bacteria. After cultivation on three commercial selective-differential media, 240 bacterial isolates were obtained and identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Isolates from clinically relevant species were further genotyped by enterobacterial repetitive intergenic consensus polymerase chain reaction, and tested for antibiotic susceptibility and presence of CPs and ESBL enzymes. Two ESBL-producing isolates and two isolates producing CPs were detected in rainwater, groundwater, and leachate: Klebsiella oxytoca complex with the gene for the ESBL enzyme CTX-M-1 and the gene for the CP OXA-48, Serratia fonticola with the gene for the ESBL enzyme FONA-2, and Pseudomonas aeruginosa with the gene coding Verona integron-encoded Metallo-beta-lactamases (VIM) metallo-beta-lactamase. Our study indicates that bacteria with ESBL and CP genes can be present in landfill-associated waters.

Analysis of gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae in rural residents of Weifang City, Shandong Province

Objective: To investigate the drug-resistant gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae (CR-ECL) in rural residents of Weifang City, Shandong Province. Methods: Fecal samples were collected from rural community residents in Weifang City, Shandong Province in 2017. Drug-resistant strains were screened using a carbapenem-resistant enterobacter chromogenic medium. CR-ECL positive strains were acquired via Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry(MALDI-TOFMS) analysis. The antibiotic resistance phenotype of CR-ECL was determined using a microbroth dilution assay. Whole genome sequencing (WGS) and analysis were conducted, along with an examination of the immediate vicinity of the blaNDM gene and phylogenetic analysis of the strains. Results: A total of 628 fecal samples were collected and tested, of which 6 were CR-ECL positive (detection rate 0.96%), all exhibiting multiple drug resistance (MDR) phenotypes. Six CR-ECL strains had four MLST genotypes (ST), all of which carried multiple drug resistance genes (blaNDM-1, blaNDM-5, etc.) and virulence genes (acrA, acrB, entB, fepC, etc.). There were mobile genetic elements ISAba125, TN3-IS3000, TN3 and IS5 in the genetic environment surrounding the blaNDM gene. The phylogenetic tree showed that the multi-locus sequence typing of the core genome (cgMLST) was consistent with the single nucleotide polymorphism (SNPs) results. The cgMLST results showed that the allele differences between strains 2BC0101B and 2BC0251B, 2BG0561B and 2BI0221B were 2 and 1, respectively. The SNPs results showed that the above two pairs of bacteria also clustered together. It was found that the strains of chicken fecal samples in the National Center for Biotechnology Information (NCBI) database were located in the center of the evolutionary tree, and the local sequences could be traced back to American human sequences. Conclusion: Multidrug-resistant CR-ECL is detected in rural community residents in Weifang City, Shandong Province.

Enrichment of phosphopeptides by arginine-functionalized magnetic chitosan nanoparticles.

One of the most crucial and prevalent post-translational modifications is the phosphorylation of proteins. The study and examination of protein phosphorylation hold immense importance in comprehending disease mechanisms and discovering novel biomarkers. However, the inherent low abundance, low ionization efficiency, and coexistence with non phosphopeptides seriously affect the direct analysis of phosphopeptides by mass spectrometry. In order to tackle these problems, it is necessary to carry out selective enrichment of phosphopeptides prior to conducting mass spectrometry analysis. Herein, magnetic chitosan nanoparticles were developed by incorporating arginine, and were then utilized for phosphopeptide enrichment. A tryptic digest of β-casein was chosen as the standard substance. After enrichment, combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the detection limit of the method was 0.4 fmol. The synthesized magnetic material demonstrated great potential in the detection of phosphopeptides in complex samples, as proven by its successful application in detecting phosphopeptides in skim milk and human saliva samples.

Distribution and antifungal susceptibility profile of oropharyngeal Candida species isolated from people living with HIV in the era of universal test and treat policy in Uganda.

Despite the increased frequency of oropharyngeal candidiasis among people living with human immunodeficiency virus (HIV), its management is no longer effective due to empirical treatment and emergence of antifungal resistance (AFR). This study sought to investigate the prevalence of oropharyngeal candidiasis and assess the antifungal susceptibility profile of oropharyngeal Candida species isolated from people living with human immunodeficiency virus. Additionally, we evaluated the correlation between oropharyngeal candidiasis and CD4 T cell as well as viral load counts. A descriptive cross-sectional study was carried out from April to October 2023 in which 384 people living with HIV underwent clinical examination for oral lesions. Oropharyngeal swabs were collected and cultured on Sabouraud Dextrose agar to isolate Candida species which were identified using the matrix assisted laser desorption ionization time of flight mass spectrometry. Additionally, the antifungal susceptibility profile of Candida isolates to six antifungal drugs was determined using VITEK® (Marcy-l'Étoile, France) compact system. Data on viral load were retrieved from records, and CD4 T cell count test was performed using Becton Dickinson Biosciences fluorescent antibody cell sorter presto. The prevalence of oropharyngeal candidiasis was 7.6%. Oropharyngeal candidiasis was significantly associated with low CD4 T cell count and high viral load. A total of 35 isolates were obtained out of which Candida albicans comprised of 20 (57.1%) while C. tropicalis and C. glabrata comprised 4 (11.4%) each. C. parapsilosis, C. dubliniensis and C. krusei accounted for 2 (5.7%) each. Additionally, 7 (20%) isolates were resistant to fluconazole, 1 (2.9%) to flucytocine and 0.2 (5.7%) isolates were intermediate to caspofungin. However, specific specie isolates like C. albicans showed 20% (4/20), C. glabrata 50% (2/4) and C. krusei 50% (1/2) resistance to fluconazole. Additionally, C. krusei showed 50% resistance to flucytosine. The prevalence of oropharyngeal candidiasis (OPC) among people living with HIV was low, and there was a significant association between OPC and CD4 T cell count as well as viral load. C. albicans was the most frequently isolated oropharyngeal Candida species. C. glabrata and C. krusei exhibited the highest AFR among the non-albicans Candida species. The highest resistance was demonstrated to fluconazole.

868. Early Organism Identification by Matrix Assisted Laser Desorption Ionization Time Of Flight Mass Spectrometry (MALDI TOF MS) Decreases the Time to Optimal Antibiotic Therapy for Common Bacterial Infections

Abstract Background MALDI-TOF helps in precise identification of microorganisms from positive cultures. It is based on the principle of proteomics and obviates the need for the use of biochemicals. It reduces the turnaround time for reporting microbiologic cultures. Early identification and reporting of the organism, before the availability of antimicrobial susceptibilities, may reduce the time to first antibiotic modification and impact the clinical outcomes of patients. Methods This is a prospective observational two-arm comparative study conducted at a tertiary care centre in North India, with a sample size of 50 patients in the conventional arm and 100 patients in the interventional arm. The workflow of both arms are depicted in figure 1&2. The study population included patients in the medical ward and intensive care units (ICU) with positive cultures. The primary objective of this study was to compare the average time to first antibiotic modification post-culture positivity in the conventional and interventional arms and to compare clinical outcomes of patients in both arms. Escalation, de-escalation and any modification in antibiotics were also recorded. Results The median time to first antibiotic modification was significantly shorter in the intervention arm compared to the conventional arm (2010 mins vs 2905 mins, p=0.002). Overall 44/100 antibiotic modifications were made in the interventional arm out of which 43.2% (19/44) were made based on the MALDI report alone without awaiting susceptibilities. The major type of modification was the de-escalation of antibiotics (47.4%). Mortality rates were 32% in the conventional arm and 27% in the intervention arm, but the difference was not statistically significant (p=0.52) (table 1). Conclusion MALDI-TOF helps in the early modification of antibiotics. The major utility is in the de-escalation of unnecessary antibiotics. Coupled with a robust antimicrobial stewardship programme, this modality can help in reducing antibiotic use. Disclosures All Authors: No reported disclosures

B-179 Development of a Reflex Mass Spectrometric Workflow for Confirmation of IgE and IgD Specificity Paraproteins Utilising the EXENT® Platform

Abstract Background The EXENT® (GAM) assay is a MALDI-TOF (Matrix Assisted Laser Desorption Ionisation Time of Flight) mass spectrometry-based solution which utilises IgG, IgA, IgM, total kappa and total lambda specific paramagnetic beads, which could be used in the investigation of monoclonal proteins in multiple myeloma and other plasma cell disorders (in development by The Binding Site (TBS), part of Thermo Fisher Scientific). Rare cases of IgD and IgE myelomas will be partially typed with the current iteration of the EXENT (GAM) assay. We present here a workflow to confirm the heavy chain specificity of such paraproteins as IgD or IgE using reflex LC-MS analysis of EXENT eluates. Methods Samples known to contain IgD (2) or IgE (3) paraproteins were provided from either routine workload at the immunology laboratory at Birmingham Heartlands Hospital, UK or an internal library of historical myeloma samples. Capillary zone electrophoresis (CZE) and immunofixation (IFE) were performed on Capillarys2™ and Hydrasys2 Scan™ platforms, respectively (Sebia, France). Serum free light chain (sFLC) measurement was performed using Freelite® reagents (The Binding Site, UK) on a Cobas c501 turbidimeter (Roche, Switzerland). Samples were prepared via EXENT (GAM) assay, by separately incubating diluted serum with antisera specific for IgG, IgA, IgM, total kappa and total lambda conjugated to paramagnetic beads. After washes, purified immunoglobulins were treated to reduce and elute the light chain component. Samples were mixed with matrix, spotted onto a MALDI target plate and acquired by MALDI-TOF MS. Once light-chain specificity of the suspected IgD or IgE paraprotein was determined, repeat immuno-precipitation with the same light-chain bead was carried out. Eluted samples were neutralised, transferred to fresh tubes, reduced, alkylated and digested with trypsin for 3 h. After acidification digested samples were analysed by C18 reverse phase UPLC on a BioAccord LC-MS system (Waters, UK). Data was then screened against a database of known immunoglobulin heavy chain peptides to assess the presence of heavy chain peptides specific for each immunoglobulin isotype. Results In each sample tested, the EXENT solution identified at least one monoclonal light chain that was only seen in the total kappa or total lambda immunoprecipitations with no corresponding monoclonal peak(s) at the same mass(es) in the IgG, IgA or IgM specificities. This finding would be consistent with the presence of clonal plasma cells producing either monoclonal free light chains only, or IgD and IgE paraproteins. Subsequent LC-MS and LC-MSe (tandem MS) analysis of reduced, alkylated and tryptically digested eluates corresponding to these suspected IgD and IgE samples produced high quality peptide sequencing data. The data identified multiple IgD or IgE specific peptides in each sample and showed between 40% and 66% sequence coverage for IgD or IgE heavy-chain constant regions. Thus, confirming the presence of IgD or IgE in these samples which had been partially typed by the EXENT solution. Conclusion This data demonstrates that the EXENT (GAM) assay could potentially aid in detection of IgD and IgE paraproteins when combined with LC-MS proteomic techniques, as it allows efficient and facile confirmation of these rare heavy chain isotypes.

Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes

Background: Foodborne infections like Listeriosis cover several disorders and are a worldwide public health emergency. Listeria monocytogenes has been isolated from various foodstuffs, including milk and fishes. MALDI-TOF mass spectrometry technique is known for its rapid and accurate identification of bacterial organisms. Methods: In the current research, a total of 360 samples comprising raw milk (130), milk products (125) and freshwater fishes (105) were screened for the detection of pathogenic Listeria species by using the USDA method. The recovered Listeria isolates were characterized using conventional set of biochemical analysis along with sugar fermentation tests and further confirmed by MALDI-TOF MS. The virulent nature of pathogenic Listeria isolates was also assessed by in vitro tests like hemolysis on blood agar, CAMP and PI-PLC assay. Result: In current study, on screening 360 animal origin food samples, three isolates were recovered from raw milk samples and identified as Listeria monocytogenes indicating an overall prevalence of Listeria monocytogenes to the tune of 0.83%. Excellent correlation was observed with identification of Listeria species using conventional phenotypic tests and advanced molecular tool Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. The results depict dependability of advanced technique for rapid and reliable identification of Listeria species.

Fc N-glycosylation of autoreactive Aβ antibodies as a blood-based biomarker for Alzheimer's disease.

Naturally occurring autoantibodies (nAbs) against the pathologic isoform of amyloid beta (Aβ42 ) were found in body fluids and indicate a systemic B cell response that may prevent Alzheimer's disease (AD) onset. N-glycans attached to immunoglobulin G-Fab/Fc fragments are features that influence their mechanism of action. The aim was to study the role of N-glycans in nAbs-Aβ42 . nAbs-Aβ42 were isolated from AD patients and age-/sex-matched controls (n = 40) and immunoglobulin preparations. Glycosylated/deglycosylated nAbs-Aβ42 were analyzed for their effect on Aβ42 's aggregation, toxicity, and phagocytosis. Glycan structure was analyzed using matrix assisted laser desorption ionization time of flight mass spectrometry. Deglycosylation of nAbs-Aβ42 had a major impact on Aβ42 's aggregation/toxicity/phagocytosis. The glycan structure showed considerable differences between AD and controls. We were able to predict disease status with a sensitivity/specificity of 95% (confidence interval [CI]: 76.4-99.7%)/100% (CI: 83.9-100%). N-glycosylation has been identified as a critical attribute maintaining the beneficial effects of autoreactive Aβ antibodies. These data have consequences for the development of monocloncal Aβ antibodies and may open new avenues for diagnostics.

Utilizing Mass Spectrometry to Detect and Isotype Monoclonal Proteins in Urine: Comparison to Electrophoretic Methods.

Matrix assisted laser desorption ionization time of flight mass spectrometry coupled to immune enrichment (MASS-FIX) as an alternative to serum immunofixation electrophoresis has demonstrated increased sensitivity in monoclonal protein (MP) detection with improved laboratory workflow. This study explored similar replacement of urine immunofixation electrophoresis (u-IFE) with urine MASS-FIX (u-MASS-FIX) by method comparison. Residual urine (n = 1008) from Mayo Clinic patients with a known plasma cell disease were assayed neat by u-MASS-FIX analysis. Each sample was paired with the following: u-IFE, urine total protein, urine protein electrophoresis, serum κ/λ free light chain (LC) ratio (rFLC), and serum MASS-FIX (s-MASS-FIX). Analytical sensitivities were measured in pooled urine spiked with daratumumab. u-IFE and u-MASS-FIX had 91% agreement in determining the presence/absence of MPs (Cohen kappa = 0.8200). In discrepant cases, serum rFLC statistically aligned more closely with positive u-MASS-FIX cases than u-IFE. Patients positive by both s-MASS-FIX and u-MASS-FIX had matching MP masses (±20 daltons) in 94% of cases. The u-MASS-FIX spectra further identified κ/λ LC fragments and glycosylated LCs not appreciated on u-IFE. The unconcentrated u-MASS-FIX limit of detection of 0.156 mg/mL was determined equivalent to 100× concentrated u-IFE. u-MASS-FIX is a reliable alternative to u-IFE with the added benefits of LC glycosylation detection and MP mass tracking between serum and urine. Furthermore, u-MASS-FIX is performed using neat urine. Eliminating the need to concentrate urine for u-IFE has potential to increase productivity by decreasing labor minutes per test.

Whole genome sequencing to study antimicrobial resistance and RTX virulence genes in equine Actinobacillus isolates

Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolates using phenotypic identification and susceptibility testing on the one hand, and long-read nanopore whole genome sequencing on the other hand. This allowed to address strain divergence down to the whole genome single nucleotide polymorphism (SNP) level. While lowest resolution was observed for 16S rRNA gene classification, a new multi-locus sequence typing (MLST) scheme allowed proper classification up to the species level. Nevertheless, a SNP-level analysis was required to distinguish A. equuli subspecies equuli and haemolyticus. Our data provided first WGS data on Actinobacillus genomospecies 1, Actinobacillus genomospecies 2, and A. arthritidis, which allowed the identification of a new Actinobacillus genomospecies 1 field isolate. Also, in-depth characterization of RTX virulence genes provided information on the distribution, completeness, and potential complementary nature of the RTX gene operons within the Actinobacillus genus. Even though overall low prevalence of acquired resistance was observed, two plasmids were identified conferring resistance to penicillin-ampicillin-amoxicillin and chloramphenicol in one A. equuli strain. In conclusion our data delivered new insights in the use of long-read WGS in high resolution identification, virulence gene typing, and antimicrobial resistance (AMR) of equine Actinobacillus species.

Pulmonary Nocardiosis Caused by <i>Nocardia otitidiscaviarum</i> and <i></i>Successfully Treated with Ciprofloxacin Based on Bacterial Identification and Antimicrobial Susceptibility Testing

We report a case of pulmonary nocardiosis caused by Nocardia otitidiscaviarum in a man diagnosed with idiopathic thrombocytopenia who was on oral prednisolone therapy. A chest PET/CT examination 13 days after the start of prednisolone therapy revealed a nodular shadow, and it progressively worsened and was accompanied by cavity formation over time. N. otitidiscaviarum was identified in bronchoalveolar lavage fluid by matrix assisted laser desorption ionization time of flight mass spectrometry. The isolate was eventually confirmed to be N. otitidiscaviarum by a 16S rRNA analysis. There was no evidence of disseminated disease. Combination therapy consisting of oral sulfamethoxazole-trimethoprim (SMX-TMP) and intravenous amikacin and imipenem was initiated, but the SMX-TMP was discontinued because of adverse effects. Antimicrobial susceptibility testing revealed that the isolate was non-susceptible to ceftriaxone, imipenem, minocycline, and clarithromycin. The patient declined long-term intravenous therapy. Oral ciprofloxacin was administered instead, and the pulmonary nocardiosis improved after 6 months of therapy. Bacterial identification and antimicrobial susceptibility testing influence the choice of treatment regimens for nocardia infections by N. otitidiscaviarum, which may be resistant to multiple antimicrobial agents.

Emergence of Clonally-Related South Asian Clade I Clinical Isolates of Candida auris in a Greek COVID-19 Intensive Care Unit.

Candida auris has recently emerged as a multidrug-resistant yeast implicated in various healthcare-associated invasive infections and hospital outbreaks. In the current study, we report the first five intensive care unit (ICU) cases affected by C. auris isolates in Greece, during October 2020-January 2022. The ICU of the hospital was converted to a COVID-19 unit on 25 February 2021, during the third wave of COVID-19 in Greece. Identification of the isolates was confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight mass spectroscopy (MALDI-TOF]. Antifungal susceptibility testing was performed by the EUCAST broth microdilution method. Based on the tentative CDC MIC breakpoints, all five C. auris isolates were resistant to fluconazole (≥32 μg/mL), while three of them exhibited resistance to amphotericin B (≥2 μg/mL). The environmental screening also revealed the dissemination of C. auris in the ICU. Molecular characterization of C. auris clinical and environmental isolates was performed by MultiLocus Sequence Typing (MLST) of a set of four genetic loci, namely ITS, D1/D2, RPB1 and RPB2, encoding for the internal transcribed spacer region (ITS) of the ribosomal subunit, the large ribosomal subunit region and the RNA polymerase II largest subunit, respectively. MLST analysis showed that all isolates possessed identical sequences in the four genetic loci and clustered with the South Asian clade I strains. Additionally, PCR amplification and sequencing of the CJJ09_001802 genetic locus, encoding for the "nucleolar protein 58" that contains clade-specific repeats was performed. Sanger sequence analysis of the TCCTTCTTC repeats within CJJ09_001802 locus also assigned the C. auris isolates to the South Asian clade I. Our study confirms that C. auris is an emerging yeast pathogen in our region, especially in the setting of the ongoing COVID-19 worldwide pandemic. Adherence to strict infection control is needed to restrain further spread of the pathogen.