MALDI-TOF MS Research Articles

Performance of Gram Stain Characteristics for Predicting Culture Growth in Lower Respiratory Specimens Across Multiple Institutions

Abstract Data from Gram stains performed on clinical specimens can guide clinical decision-making and downstream laboratory workflows. However, the extent to which Gram stain results predict gold-standard culture results, and the generalizability of this performance across sites, warrants additional exploration. We sought to discover which Gram stain features would be associated with clinically significant culture growth (“growth”). To do so, 574 remnant specimens from the lower respiratory tract collected from four institutions were analyzed. A standardized Gram stain and culture protocol was performed across all study sites. Final reference organism identification was assigned using MALDI-ToF MS, and clinically significant growth was defined as any organism reported to the medical record. Gram stains with mixed microorganisms detected were considered concordant if ≥2 organisms of different Gram stain appearances were identified in culture. Growth was observed in 53% of specimens, including 25% of specimens in which Gram stain demonstrated no organisms. The 95% confidence intervals (CI) of the site-wise growth rates were 30-42%, 42-52%, 51-67%, and 60-77% (p < 0.001 by Chi-squared). By Gram stain morphology, 53-69% of specimens with Gram-positive cocci grew any clinically significant bacteria, while 31-49% grew a concordant Gram-positive cocci. 65-85% of specimens with Gram-negative bacilli grew any clinically significant bacteria, while 59-74% grew a concordant Gram-negative bacilli. 63-83% of specimens with mixed microorganisms grew 2 or more clinically relevant bacteria of differing Gram stain morphologies. Specimens from bronchoalveolar lavages (BALs) were less likely to have growth (30-42%) than endotracheal aspirates (66-77%). Polymorphonuclear leukocyte (PMNs) abundance was associated with culture growth, with Gram stains showing no, rare, few, moderate, or abundant PMNs demonstrating growth 28, 44, 52, 60, and 67% of the time (p < 0.001). Specimens with None and few squamous epithelial cells on Gram stain demonstrated growth 38-52% and 59-68% of the time (p < 0.001). No significant difference in growth was observed for red blood cells. Organism abundance on Gram stain was significantly associated with culture growth, with growth rate CIs being 20-32% for no organisms, 44-62% for rare, 58-77% for few, 70-93% for moderate, and 81-95% for abundant organisms seen. The extent to which these associations could guide the development of decision rules or machine learning models that demonstrate acceptable negative predictive value for pathogen growth to alleviate the operational burden of low-utility cultures is an exciting future direction to explore.

Long Stability of Atomically Precise Red Emissive Copper Nanoclusters within the Gel and Their Use As a Potential Catalyst and Fluorescent Ink.

Herein, an amphiphile-based hydrogel (with 5% DMF) containing natural amino acid residue has been used to prepare and stabilize red-emitting CuNCs for several months. Though different methods have been attempted, amphiphile and 4-mercaptobenzoic acid (4-MBA)-containing hydrogels are pinpointed to be the base medium to stabilize this new Cu-cluster. From a MALDI-TOF MS analysis it was found that it is a Cu8-atom cluster stabilized by three 4-MBA ligands. Copper acetate monohydrate (Cu(CH3COO)2·H2O) has been used as a copper precursor, and l-ascorbic acid has been used as a reducing agent. FEG-TEM analysis shows that the Cu cluster has an average size of 2.83 nm. Interestingly, these clusters can be used as a fluorescent ink with a visibility of the solid state under a UV-lamp with an excitation of 365 nm. This envisaged applying these CuNCs for anticounterfeiting. These Cu-clusters show an excitation of 420 nm with an emission of 620 nm, as is evident from the fluorescence spectroscopic analysis. Based on our knowledge, this is the first example of making and consequently stabilizing Cu-clusters using hydrogel as a template for a few months. Moreover, these CuNCs can also be used as a catalyst for the reduction of nitro derivatives to their amine derivatives in aqueous medium.

Determination of nut varieties and their detection in festive cookies by MALDI-TOF mass spectrometry.

Nuts contain a large amount of essential fatty acids, amino acids, and whole range of minerals and vitamins valuable for human health, yet certain risks are associated with their consumption, of which allergic reaction is the most important. Considering the growing number of people suffering from allergies caused by allergens of protein origin, the aim of this work is to find out whether nuts can be distinguished from each other on the basis of contained proteins. A total of eleven raw and subsequently heat-treated nuts (almonds, Brazil nuts, cashews, coconuts, hazelnuts, macadamia nuts, peanuts, pecans, pine nuts, pistachios, and walnuts) were analyzed using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry with the subsequent finding of characteristic m/z values for each analyzed nut. No previous method for protein extraction was used. The characteristic values were used to verify the composition of seven types of festive cookies - six commercial products and one "unknown" cookie, where it was not known in advance, which nut it was made from. The procedure, together with the found characteristic m/z values, could serve to rapidly identify the plant origin of nut products.

MALDI-TOF MS profiling to predict resistance or biofilm production in gram-positive ESKAPE pathogens from healthcare-associated infections

Antimicrobial resistance and biofilm production in healthcare-associated infections is a health issue worldwide. This study aimed to identify potential biomarker peaks for resistance or biofilm production in ESKAPE pathogens using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility and biofilm production were assessed on selected isolates. Biomarker peaks were identified using MALDI Biotyper and ClinProTools software. Among resistant strains, 90.0 % were carbapenem-resistant Acinetobacter baumannii (CRAB), 39.0 % were methicillin-resistant Staphylococcus aureus (MRSA), 17.98 % were multidrug-resistant (MDR) Pseudomonas aeruginosa, 21.6 % were vancomycinresistant Enterococcus (VRE), and 2.55 % were carbapenem-resistant Enterobacterales (CRE). Biofilm production was 40.0 % in VRE and 45.8 % in MRSA. Although no potential biomarker peaks for biofilm production were detected, several potential biomarker peaks for drug resistance in VRE (n=5), MRSA (n=4), and MDR P. aeruginosa (n=4) were detected, suggesting avenues for the development of rapid diagnostic tools.

Identification of cotton non-pathogenic fungal agent isolates based on morphological and MALDI-TOF mass spectrometry method

In this study, morphological and MALDI-TOF MS identification and comparison of non-pathogenic fungal species isolated from diseased root, leaf and boll tissues of cotton plants were carried out. For this purpose, surveys were conducted in Bağlar, Sur, Çınar, Bismil, Yenişehir, Ergani, Eğil, Kayapınar and Silvan districts of Diyarbakır province where cotton production is intensive between June and September 2020 and 2021. 209 samples of plants showing typical fungal disease symptoms were collected from 75 different cotton production areas. A total of 171 fungal isolates were obtained by isolation, culture and purification procedures from diseased plant tissues in the samples. The 20 isolates that were negative in the pathogenicity test in the main host were identified and compared by morphological (traditional) and MALDI-TOF MS methods. According to the results, Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Aspergillus fumigatus, Aspergillus minisclerotigenes, Penicillium nalgıovense, Chaetomium globosum, Dichotomopilus funicola, Arthroderma gloria, Pseudogmnoacus pannorum, Tichophyton interdigitale, and Penicillium sp. Penicillium sp. were found to be intensively colonized in different parts of cotton such as leaves, bolls and roots. Again, it was determined that the most common species among the total saprophyte isolates was Aspergillus niger with a high similarity rate.

Antiproliferative Effects of Naja anchietae and Naja senegalensis Venom Peptides on Glioblastoma Cell Lines.

This study explores the potential of natural bioactive peptides from animal venoms as targeted anti-cancer agents with reduced toxicity. Initially, we screened a broad collection of animal venoms for their antiproliferative activity against cancer cell lines. From this collection, we selected venoms from Naja anchietae and Naja senegalensis due to their promising activity. Utilizing reverse- phase high-performance liquid chromatography (RP HPLC), mass spectrometry (MALDI-TOF MS and MALDI-TOF TOF MSMS), and Edman degradation sequencing, we isolated and characterized three peptides named CTNanc1, CTNanc2, and CTNanc3 from Naja anchietae, and three others named CTNsen1, CTNsen2, and CTNsen3 from Naja senegalensis, each with a molecular weight of around 7 kDa. These purified peptides demonstrated inhibition of U87 glioblastoma cell proliferation, but not of U251 and T98G cells, in cell viability assays. To assess the impact of these treatments on cell viability, apoptosis, and necrosis, flow cytometry assays were conducted on U87 cells at 72 h. The results showed a decrease in cell viability and an increase in dead cells, suggesting that the treatments not only promote apoptosis, but may also lead to increased necrosis or late-stage apoptosis as the exposure time increases. These findings suggest that these peptides could be developed as leads for cancer therapy.

Synthesis of a dehydrodieugenol B derivative as a lead compound for visceral leishmaniasis-mechanism of action and in vivo pharmacokinetic studies.

Leishmaniasis is a parasitic neglected tropical disease, affecting 12 million people. Available treatments present several limitations, with an increasing number of resistance cases. In the search for new chemotherapies, the natural product dehydrodieugenol B was used as a scaffold for the synthesis of a series of derivatives, resulting in the discovery of the promising analog [4-(4-(5-allyl-3-methoxy-2-((4-methoxybenzyl)oxy)phenoxy)-3-methoxybenzyl)morpholine, 1]. In this work, we investigated the effect of compound 1 on cell signaling in Leishmania (L.) infantum, culminating in cell death, as well as its immunomodulatory effect in the host cell. Additionally, we performed a pharmacokinetic profile study in an animal model. After treatment, compound 1 induced the alkalinization of acidocalcisomes and concomitant Ca2+ release in the parasite. These events may induce depolarization of the mitochondrial potential, with successive collapse of the bioenergetic system, leading to a reduction of ATP and reactive oxygen species (ROS) levels. The analysis of total proteins and protein profile by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) demonstrated that compound 1 also altered the parasite proteins after treatment. Transmission electron microscopy studies revealed ultrastructural damage to mitochondria; together, these data suggest that compound 1 may promote autophagic cell death. Additionally, compound 1 also induced an immunomodulatory effect in host cells, with a reduction of Th1 and Th2 cytokine response, characterizing an anti-inflammatory compound. The obtained pharmacokinetic profile in rats enhances the potential of the compound, with a mean plasma half-life (T1/2) of 21 h. These data reinforce the potential of compound 1 as a new lead for future efficacy studies.

Evaluation of protein extraction protocols for MALDI-TOF Biotyper analysis of mycobacteria

Infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of M. bovis BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: M. peregrinum, M. smegmatis, and M. farcinogenes. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories.

Successful treatment of Keratitis caused by Mycobacterium chelonae and an overview of previous cases in Europe

Introduction and purposeMycobacterium (M.) chelonae is responsible for a half of relatively rare nontuberculous mycobacteria (NTM) keratitis. We report a case of M. chelonae keratitis in a woman following sclerocorneal suture extraction after cataract surgery.ResultsA 70-year-old woman presented with a red eye and corneal infiltration of her left eye six weeks following sclerocorneal suture extraction after an elective cataract surgery in another institute. She complained of a sharp, cutting pain and photophobia. Since initial corneal scrapes and conjunctival swabs proved no pathogen using culture and PCR methods, non-specific antibiotics and antifungal agents were administered. As keratitis was complicated by an inflammation in the anterior chamber and vitreous, samples of the vitreous fluid were sent for microbiologic examination. DNA of Epstein-Barr virus (EBV) was repeatedly detected. Since the intrastromal abscess had formed, corneal re-scrapings were performed and M. chelonae was detected using culture, MALDI-TOF MS and PCR methods. Therapy was changed to a combination of oral and topical clarithromycin, intravitreal, topical and intracameral amikacin, and oral and topical moxifloxacin. The successful therapy led to stabilization. The optical penetrating keratoplasty was performed and no signs of the infection recurrence were found.ConclusionsThe diagnosis of nontuberculous mycobacterial keratitis is difficult and often delayed. An aggressive and prolonged antimicrobial therapy should include systemic and topical antibiotics. Surgical intervention in the form of corneal transplantation may be required in the active and nonresponsive infection. In the presented case this was necessary for visual rehabilitation due to scarring.

A convenient and versatile culturomics platform to expand the human gut culturome of Lachnospiraceae and Oscillospiraceae.

The human gut microbiota is increasingly being recognised to play an important role in maintaining health. The families Lachnospiraceae and Oscillospiraceae in particular, are often reduced in disease states but are relatively poorly represented in culture collections. Cultured representatives are required to investigate the physiology and host interactions of gut microbes. Establishing cultured isolate collections can be laborious and expensive owing to the fastidious growth requirements of these organisms and the costs associated with taxonomic classification. This study proposes a culturomics platform combining a single basal culture medium with matrix-assisted laser adsorption/ionisation coupled to time-of-flight mass spectrometry (MALDI-TOF MS) for fast and reliable isolation and identification of hundreds of novel isolates. In this study, basal YCFA medium supplemented with either glucose, apple pectin, or porcine mucin was used to cultivate a total of 724 different isolates derived from only 11 different faecal samples from healthy volunteers, of which 389 isolates belonged to the Lachnospiraceae and Oscillospiraceae families. Moreover, 27 isolates could not be assigned to known species based on their 16S rRNA gene, 17 of which may even represent novel genera. To aid MALDI-TOF MS identification of gut bacteria, the commercial database was complemented with the MaldiGut database presented here, containing a collection of 132 different Main Spectrum Profiles, including the profiles of 125 Firmicutes species, 3 Bacteroidetes species, 3 Actinobacteria species, and one Verrucomicrobia species. The culturomics platform and MaldiGut database presented here will enable further expansion of the gut culturome, especially within the understudied Lachnospiraceae and Oscillospiraceae families.

Oriental covalent immobilization of N-glycan binding protein via N-terminal selective modification

Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man3GlcNAc2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability.

Characterization of neurologic disease-associated Streptococcus suis strains within the United States swine herd and use of diagnostic tools.

Streptococcus suis negatively impacts swine health, posing diagnostic and preventative challenges. S. suis can induce disease and also quietly reside on mucosal surfaces. The limited use of diagnostic tools to identify disease-associated strains and rule out differential diagnoses, alongside the complex ecology of S. suis, poses significant challenges in comprehending this important pathogen and defining pathotypes. This study evaluated 2,379 S. suis central nervous system (CNS) isolates from diagnostic submissions between 2015 and 2019. Isolates originating from submissions with histologic evidence of CNS infection (n = 1,032) were further characterized by standard and advanced diagnostic techniques. We identified 29 S. suis serotypes and 4 reclassified serotypes as putative causes of CNS disease. Among these, serotypes 1 and 7 emerged as the predominant putative causes of CNS infection (32% of submissions). Furthermore, 51 sequence types (STs), of which 15 were novel, were detected with ST1 predominating. Through whole-genome sequencing of 145 isolates, we observed that five commonly used virulence-associated genes (VAGs; epf, mrp, sly, ofs, and srtF) were not present in most disease-associated isolates, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) yielded false-positive results in 7% of isolates. These data indicate that (i) clinical signs and site of isolation alone are insufficient for defining a pathotype, (ii) S. suis serotypes and STs associated with CNS infection are more diverse than previously reported, (iii) MALDI-TOF MS may need to be supplemented with additional diagnostic tools for precise S. suis identification, and (iv) VAGs remain an unreliable means for identifying isolates associated with CNS disease.IMPORTANCEStreptococcus suis is an important and complex systemic bacterial pathogen of swine. Characterization of S. suis strains originating from pigs with histologic confirmation of neurologic disease is limited. Review of swine diagnostic submissions revealed that fewer than half of cases from which S. suis was isolated from the brain had histologic evidence of neurologic disease. This finding demonstrates that clinical signs and site of isolation alone are not sufficient for identifying a neurologic disease-associated strain. Characterization of strains originating from cases with evidence of disease using classic and advanced diagnostic techniques revealed that neurologic disease-associated strains are diverse and commonly lack genes previously associated with virulence.

Genotypic and phenotypic characterization of the first Latin America isolates of Corynebacterium rouxii, a recently described member of the Corynebacterium diphtheriae complex reported in Europe.

The genus Corynebacterium is the largest genera among corynebacteria and has a range of species widely spread in ecological niches, some with epidemic potential and capable of causing fatal diseases. In recent years, due to the reclassifications and discoveries of new potentially toxin-producing species, microbiological identification and epidemiological control have been compromised, becoming possible only with sequencing techniques. Two bacterial strains isolated from a cat were identified by MALDI-TOF mass spectrometry as Corynebacterium diphtheriae and sent to the collaborating center of the Brazilian Ministry of Health for molecular identification and determination of toxigenicity potential, which were initially performed by multiplex PCR method. In addition, the antimicrobial susceptibility profile was determined according to BrCAST. Finally, for the final identification at the species level and effective epidemiological monitoring, the sequencing of the 16S rRNA and rpoB housekeeping genes was carried out. The isolates were identified as nontoxigenic C. diphtheriae strains by mPCR. Both strains were found susceptible to all antimicrobial agents. Although the identification at the species level was not possible through similarity analysis of S rRNA and rpoB housekeeping genes, the phylogenetic analysis showed that the isolates belonged to the species Corynebacterium rouxii with a high value of reliability. This is the first report of the isolation of C. rouxii in Latin America. Molecular identification, whether by the MALDI-TOF mass spectrometry or PCR techniques, does not discriminate C. rouxii from C. diphtheriae, requiring gene sequencing and phylogenetic analysis for correct identification at the species level.

The Diversity of N-Glycans of Chlorella Food Supplements Challenges Current Species Classification.

N-glycans have recently emerged as highly varied elements of Chlorella strains and products. Four years and eighty samples later, the increasing N-glycan diversity calls for a re-examination in the light of concepts of species designations and product authenticity. N-glycans of commercial products were analyzed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF MS) supported by chromatography on porous graphitic carbon with mass spectrometric detection. Although 36% of 172 products were labeled C. vulgaris, only 9% presented what could be taken as a C. vulgaris type N-glycan pattern. Respectively, 5 and 20% of the products matched with C. sorokiniana strains SAG 211-8k and SAG 211-34, which, however, carry entirely different structures. Furthermore, 41% presented with one of four frequently occurring glyco-types while 26% of the samples showed unique or rare N-glycan patterns. These glycan signatures thus profoundly challenge the stated species designations. By no means do we want to question the presumed health benefits of the products or the sincerity of manufacturers. We rather aim to raise awareness of the fascinating but also concerning diversity of microalgal N-glycans and suggest it as a means for defining product identity and taxonomic classifications.

Antimicrobial Action of Lactobacillus spp. Isolated from Yoghurt against Escherichia coli, Salmonella Enteritidis and Listeria monocytogenes: A Pilot Study

Milk has been a dietary staple around the world for centuries. In recent years, consumer interest in healthy foods and organic products has increased due to their health-promoting properties. Fermented dairy products, including yoghurt, are receiving special attention for their properties and the presence of probiotic bacteria. The quantitative and qualitative (MALDI TOF MS) evaluation of lactic acid bacteria (LAB) in different types of yoghurt (with different shelf lives) was carried out. The effect of the Lactobacillus spp. strains isolated from yoghurts (with potential antimicrobial activity) against foodborne pathogenic bacteria (Escherichia coli, Salmonella Enteritidis and Listeria monocytogenes) was evaluated. The presence of Lactobacillus spp. (Lactobacillus rhamnosus and Lactobacillus paracasei) in the tested yoghurts was demonstrated. In the samples tested, not all the lactic acid bacteria (LAB) declared by the manufacturer were identified. The number of live bacteria present in the product was influenced by the type of yoghurt. The number of bacteria did not fall below the World Health Organization (WHO) recommended level by the last day of validity. It was shown that a mixed culture (L. rhamnosus and L. paracasei, isolated from tested yoghurts) had the most significant effect on changing the number of pathogenic microorganisms. The consumption of dairy products, which are a source of LAB, can reduce the risk of foodborne pathogen infections.

Analysis of the changes of bacterial spectrum and drug resistance in sputum culture of ICU children in a hospital of pediatric in Jiangsu Province from 2017 to 2022

Objective: To investigate the changes of the distribution and drug resistance profile of bacteria from ICU children with lower respiratory tract infection (LRTI) in Suzhou City, Jiangsu Province from 2017 to 2022. Methods: From January 2017 to December 2022, a cross-sectional observational study on the bacterial spectrum analysis among intensive care unit (ICU) children with LRTI was conducted in Children's Hospital of Soochow University. The bacteria was cultivated by culture methods from sputum samples, and identified by MALDI-TOF mass spectrometry. Drug sensitivity tests were performed by the VITEK2 Compact fully automated analysis system and the paper slide method. The χ2 test or Fisher's exact probability was used to analyze the changes of the distribution of sputum culture-positive bacteria and drug resistance in ICU children. Results: The overall detection rate of sputum culture was 42.06% (1 182/2 810). Staphylococcus aureus (25.63%,303/1 182), Acinetobacter baumannii (13.62%,161/1 182) and Haemaphilus influenzae (13.28%,157/1 182) were the top three. Proportions of Acinetobacter baumannii (17.90% vs. 11.02%,χ²=11.17, P=0.001), especially carbapenem-resistant Acinetobacter baumannii (43.70% vs. 23.50%, χ²=15.21, P<0.001) increased significantly from 2020 to 2022. However, the proportions of Haemophilus influenzae (8.50% vs. 16.19%, χ²=14.27, P<0.001), Streptococcus pneumoniae (8.50% vs. 15.92%, χ²=13.42, P<0.001) and extended-spectrum-lactamase producing Escherichia coli (8.89% vs. 18.00%, χ²=5.45, P=0.025) decreased. Drug resistant results showed that Acinetobacter baumannii was obviously more resistant to imipenem (χ²=4.43, P=0.035) and levofloxacin (χ²=12.53, P<0.001), while more sensitive to minocycline (χ²=8.34, P=0.004). Escherichia coli showed a significant increase in resistance to piperacillin tazobactam (χ²=8.29, P=0.008) and cefoperazone sulbactam (χ²=5.07, P=0.024) from 2020 to 2022; Klebsiella pneumoniae consistently maintained a resistance rate of more than 60% to first and second-generation cephalosporins, and remain susceptible to quinolones and carbapenems. Staphylococcus aureus remained highly susceptible to levofloxacin (drug resistance rate: 2.31%,7/303) and sulfamethoxazole/trimethoprim (drug resistance rate: 4.95%,15/303) from 2020 to 2022. Conclusion: Higher detection and resistance rates of Acinetobacter baumannii from sputum culture in ICU children from 2020 to 2022 were explored. Resistance of Escherichia coli to β-lactamase inhibitor combinations was more serious. Regular monitoring the changes of the etiology of respiratory tract infections in ICU Children is particularly important for the prevention and treatment of multidrug-resistant bacterial infections.

Ultrafast metaproteomics for quantitative assessment of strain isolates and microbiomes

BackgroundMicrobial communities are essential in human health and environmental regulation, but present a challenge for the analytical science due to their diversity and dynamic range. Tandem mass spectrometry provides functional insights on microbial life cycle, but is time-consuming. MALDI TOF excels in rapid species identification, but not functional assessment. To address critical challenges in human health and environmental sustainability, microbiology needs advanced mass spectrometry methods and bioinformatic tools enabling both rapid identification and accurate assessment of functional activity of microbial communities. ResultsWe show for the first time that both identity and functional activity of microorganisms and their communities can be accurately determined in experiments as short as 7 min per sample, using the basic Orbitrap MS configuration without peptide fragmentation. The approach was validated using strain isolates, mock microbiomes composed of bacteria spiked at known concentrations and human fecal microbiomes. Our new bioinformatic algorithm identifies the bacterial species with an accuracy of 95 %, when no prior information on the sample is available. Microbiome composition was resolved at the genus level with the mean difference between the actual and identified components of 12 %. For mock microbiomes, Pearson coefficient of up to 0.97 was achieved in estimates of strain biomass change. By the example of Rhodococcus biodegradation of n-alkanes, phenols and its derivatives, we showed the accurate assessment of functional activity of strain isolates, compared with the standard label-free and label-based approaches. SignificanceOur approach makes microbial proteomics fast, functional and insightful using the Orbitrap instruments even without employing peptide fragmentation technology. The approach can be applied to any microorganisms and can take a niche in routine functional assessment of microbial pathogens and consortiums in clinical diagnostics together with MALDI TOF MS and 16S rRNA gene sequencing.

6644 Glycosylation Patterns of the IgG Variable Region in Thyroglobulin Antibodies and Their Impact on Antigen Binding Affinity

Abstract Disclosure: S. Wang: None. Y. Cao: None. C. Zhao: None. C. Huang: None. K. Zhao: None. Y. Li: None. Y. Huang: None. Z. Ying: None. Y. Zhang: None. Y. Gao: None. Objective: Thyroglobulin antibodies (TgAb), key serological markers in Hashimoto's thyroiditis (HT) patients, are predominantly in immunoglobulin G (IgG) form. Prior research has highlighted the importance of increased glycosylation in TgAb IgG from HT patients. This study investigates the unique glycosylation patterns of F(ab')₂ fragments in TgAb IgG in HT patients, compared to healthy individuals. It focuses on how these patterns differ and their impact on binding to thyroglobulin (Tg). Exploring the role of N-glycosylation in the F(ab')₂ segment of TgAb IgG is crucial for understanding its effect on immune responses in HT, potentially unveiling key mechanisms of the disease. Methods: To assess serum TgAb levels in HT patients (n≥28), we used an electrochemiluminescence immunoassay, dividing patients into medium-level (mHT, n≥14) and high-level (hHT, n≥14) groups. TgAb IgG was isolated from serum of both HT and control groups (n = 14) using affinity chromatography. TgAb IgG was then cleaved with Ides enzyme to yield TgAb IgG F(ab’)2 and Fc fragments. The F(ab')2 fragment of TgAb IgG underwent MALDI-QIT-TOF-MS mass spectrometry for glycosylation profiling. Additionally, mixed TgAb IgG was separated using columns that recognize mannose and terminal sialic acid, yielding high mannose and high terminal sialic acid TgAb IgG. Different TgAb IgG glycoforms were prepared through enzymatic treatment with peptide-N-glycosidase F, β1-4 galactosidase, and α2-3,6,8 neuraminidase. The binding affinity of these TgAb IgG to Tg antigen was measured using Tg specific ELISA and surface plasmon resonance (SPR). Results: Using MALDI-TOF MS, we identified 22 IgG glycan types in both HT and control (Con) groups. In the hHT group, six glycan types (G0F, G1F, G2F, M5A1, A3G1F, A3F) were upregulated compared to Con, with statistical significance (p &amp;lt; 0.05). The mHT group showed increased levels of G1F and G2F compared to Con (p &amp;lt; 0.05). Between hHT and mHT, only A3F was significantly elevated (p &amp;lt; 0.05). Removal of N-glycosylation from IgG TgAb F(ab)2 reduced binding to Tg antigen. Presence of both mannose and terminal sialic acid in TgAb enhanced the binding to Tg. Conclusions: Most TgAb IgG in the variable region of HT patients contain N-glycans. The glycosylation patterns of TgAb IgG F(ab)2 in HT differ significantly from healthy controls, affecting antibody binding affinity. Our findings reveal the complex role of glycosylation patterns in TgAb IgG, providing new insights into HT pathogenesis and potential research directions. Presentation: 6/1/2024

Utilization of MALDI-TOF MS in the etiological diagnosis of deep-seated anaerobic bacterial infections

PurposeDeep-seated abscesses can be caused by a wide array of bacteria in various anatomical sites, the precise identification of which is crucial for implementing organism-specific treatments which can reduce morbidity and mortality. MALDI-TOF MS is a powerful proteomic method for the swift and accurate identification of anaerobic organisms. The aim of this study was to investigate deep-seated infections by MALDI-TOF MS (in comparison to VITEK®2 ANC ID card and phenotypic biochemical tests) and to determine the susceptibility pattern of identified microorganisms. Materials and methodsA total of 104 samples from patients suspected of deep-seated infections were aseptically collected and subjected to microscopy, aerobic/anaerobic cultures and subsequent identification via MALDI-TOF MS followed by antimicrobial susceptibility testing. Anaerobic bacteria were also identified using the VITEK-2 system and phenotypic biochemical tests. ResultsOut of the 104 samples tested, 41.3 % (43/104) showed positive results, predominantly in pus specimens (88 %). Mixed infections were found in 21 % of the positive cases. Of the 52 organisms identified from positive specimens, 19.2 % (10/52) were obligate anaerobes, with Bacteroides fragilis group being the most prevalent, followed by both Clostridium perfringens and Clostridium sporogenes respectively. Escherichia coli was observed to be the most common facultative anaerobic isolate. All obligate anaerobes were successfully identified to the species level via MALDI-TOF MS. In contrast, the VITEK®2 ANC ID card identified only 40 % (4/10) anaerobic bacteria to the species level. All obligate anaerobic organisms showed 100 % susceptibility to metronidazole, vancomycin and ertapenem. 25 % of the Bacteroides spp. and 50 % of Clostridium perfringens isolates were found to be resistant to clindamycin. ConclusionMALDI-TOF MS proves as a beneficial diagnostic tool for bacterial identification, eliminating the labour-intensive and time consuming conventional microbiological methods. Its accuracy of bacterial detection further helps in combating antibiotic resistance and improving patient outcomes in deep-seated infections.

A rare case report of tissue infection caused by Pantoea piersonii (basionym Kalamiella piersonii).

In 2019, Pantoea piersonii was initially isolated from the interior surfaces of the International Space Station. This microorganism is a species within the genus Pantoea in the family Erwiniaceae, belonging to the order Enterobacterales. Recent literature has documented four cases of its isolation. Despite initial predictions suggesting the non-pathogenicity of P. piersonii strains, evidence from observed cases indicates potential pathogenicity. According to documented evidence in the literature, this microorganism is capable of causing severe and life-threatening conditions, including sepsis. Traditional tests, as well as automated systems, may fail to provide complete differentiation due to these similarities. While MALDI-TOF MS is a valuable tool for identification in clinical diagnostic microbiology, sequencing may be necessary for precise identification. To determine the antibiotic susceptibility profile, various methods can be utilized, including minimum inhibitory concentration determination, disk diffusion testing (Kirby-Bauer test), genotypic resistance assays (PCR and sequencing), and automated systems. The literature reports a limited number of cases associating P. piersonii with human infection. This study contributes to this body of knowledge by reporting a novel case in which P. piersonii was isolated from a tissue sample for the first time. In this case report, the patient achieved recovery following the administration of appropriate antibiotic treatment based on the diagnosis. It underscores the need for precise identification and understanding of its pathogenicity.