MALDI-TOF MS Research Articles

The crucial quality marker of Panax ginseng: Glycosylated modified ribonuclease-like storage protein

Panax ginseng C.A.Mey is a famous natural herbal medicine worldwide. Mountain-cultivated ginseng (MCG) and garden-cultivated ginseng (GCG) are two types of Panax ginseng. There is a significant difference in economic benefits between MCG and GCG, which can always lead to problems such as adulteration and substitution of MCG with lower-priced alternatives. We explored the quality marker of ginseng at the intact protein level and established a foundation for the quality control of ginseng. Cellulose nanocrystal assisted sample preparation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) equipped with a high mass detector was performed to analyze intact proteins in ginseng. The results revealed that the ribonuclease-like storage protein is the most abundant protein in MCG and GCG. Meanwhile, the molecular weight of the ribonuclease-like storage protein showed great difference between different ginseng species, which is 26.2 kDa in MCG and 24.2 kDa in GCG. The ribonuclease-like storage protein glycosylation modification difference provides data support for the differentiation between MCG and GCG. This study showed that glycosylated modified ribonuclease-like storage protein can be a crucial quality marker of ginseng, facilitating the rapid distinction between MCG and GCG.

A Day Saved is a Life Saved: Direct Antimicrobial Susceptibility Testing from Positively Flagged Blood Culture Bottles and their Concordance with the Routine Method.

Sepsis is a major health problem worldwide and is associated with high morbidity and mortality with every hour delay in initiation of therapy. A conventional method of blood culture and Antimicrobial Susceptibility Testing (AST) takes around 48-72 hours. Empirical antibiotics need to be administered until the sensitivity report is made available. It has been estimated that 20-50% of the empirical antibiotics are inappropriate, resulting in prolonged hospital stays, adverse effects, and emergence of drug resistance. Additionally, this also puts an extra financial burden on both the patients and healthcare settings. Performing direct Antimicrobial Sensitivity Testing (dAST) is an important tool to reduce turn-around time (TAT) by at least 18-24 hours, thus reducing morbidity and mortality among critically ill patients. Direct AST (dAST) was performed from the positively flagged blood culture bottles received between December, 2021 to May, 2022 from Intensive Care Units (ICUs) on Mueller- Hinton Agar (MHA) using four drops of withdrawn blood. dAST was performed for six drugs: Ceftriaxone-30 μg (CTR), Piperacillin/Tazobactam-100/10 μg (PIT), Meropenem-10 μg (MRP), Ciprofloxacin-5 μg (CIP), Aztreonam-30 μg (AT), and Colistin (CL). The zone of inhibition was interpreted as per CLSI M100 ed32, 2022 guidelines. A parallel conventional method was also performed to examine for categorical agreement and disagreement. Identification was carried out using MALDI-TOF MS from the colonies that appeared on the dAST plate on the subsequent day. A total of 162 positively flagged blood culture bottles were included in the study. The majority of the Gram-negative organisms were from Enterobacterales (n=109), followed by Acinetobacter spp. (n=28) and Pseudomonas aeruginosa (n=25). Out of the 972 isolate-antimicrobial combinations, overall Categorical Agreement (CA) was seen in 936 (96.3%), whereas disagreement was observed in 36 with minor error (mE) in 21 (2.2%), major error (ME) in 7 (0.7%), and very major error (VME) in 8 (0.8%) when compared to the routine method. Categorical agreement (CA) of > 99% was seen in ceftriaxone (CTR) and ciprofloxacin (CIP). In comparison, the lowest CA was observed with meropenem (MRP) at 92%. Colistin dAST was performed using the E-strip method, and the result obtained was highly convincing, with an overall disagreement of only 1.2%. Rapid dAST from positively flagged blood culture bottles proved to significantly reduce the TAT from the time of sample collection to the first availability of antimicrobial susceptibility report with excellent categorical agreement of > 95% using the conventional disc diffusion method. Results obtained were within the acceptance criteria set by U. S. Food and Drug Administration (FDA) guidelines of > 90% categorical agreement for a new method. We were able to obtain excellent concordance for colistin using the E-strip method. Performing dAST not only saves a "day", but its proper implementation would save a "life".

Cyclopeptide Avellanins D-O with Antimalarial Activity from the Mariana Trench Anemone-Derived Hamigera ingelheimensis MSC5.

Marine microorganisms are a treasure trove of natural products, especially those in extreme marine environments, which may produce novel natural products. Herein, biosynthetic gene cluster analysis combined with an integrated metabolomic strategy incorporating matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) based Global Natural Products Social Molecular Networking (GNPS) was used to discover new compounds from the Mariana trench anemone-derived fungus Hamigera ingelheimensis MSC5. As a result, 12 new cyclic pentapeptides, avellanins D-O (1-12), were isolated, together with a known cyclic pentapeptide avellanin C (13). All the structures and absolute configurations were elucidated using NMR, mass spectrometry, X-ray diffraction analysis, and Marfey's method. A plausible biosynthetic pathway for the avellanins was proposed based on the gene cluster analysis of H. ingelheimensis MSC5. Bioassay revealed that compound 6 exhibited potent antimalarial activity with an IC50 value of 0.19 ± 0.09 μM.

The neurotoxic effect of Naja nubiae (Serpentes: Elapidae) venom from Sudan.

Neurotoxicity is a common feature of elapid snake envenomation. There are limited studies on the toxicity of Naja nubiae venom, the Nubian spitting cobra, from north-east Africa. We used the chick biventer cervicis nerve-muscle preparation to demonstrate the neurotoxic effect of N. nubiae venom and to compare it with the potent neurotoxic cobra Naja melanoleuca venom. Venoms were separated by successive reverse-phase high-performance liquid chromatography (RP-HPLC) runs and the molecular mass of the neurotoxins was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Both venoms caused time-dependent inhibition of nerve-mediated twitches with a t90 value of 22.2±1.9min and 12.9±1.2min for N. nubiae and N. melanoleuca venoms, respectively. Prior incubation of some commercial antivenom (EchiTab-Plus-ICP [Costa Rica], CSL, Parkville, Victoria, Australia) and snake venom antisera [India]) did not prevent the neurotoxic effect of N. nubiae venom. The chromatographic separation of N. nubiae and N. melanoleuca venoms followed by MALDI-TOF MS analysis revealed that short-chain α-neurotoxin accounted for 8.4% of N. nubiae and 14.8% of N. melanoleuca whole venoms. N. nubiae venom, which was previously known as cytotoxic venom, exhibits considerable in vitro neurotoxic effects on chick nerve-muscle preparations that may have consequences for antivenom development in north-east Africa.

Assessing Normandy Soil Microbial Diversity for Antibacterial Activities Using Traditional Culture and iChip Methods

Uncultured microorganisms represent a promising and untapped source of antibacterial compounds, crucial in the fight against the significant threat of antimicrobial resistance (AMR). In this study, both traditional and isolation chip (iChip) cultivation techniques were employed to enhance the recovery of known and unknown microorganisms from soils located in Normandy, France. The isolates obtained were identified using 16S rDNA or ITS regions analysis and MALDI-TOF mass spectrometry and were screened for antibacterial activity. A total of 386 isolates, belonging to 6 microbial phyla and distributed across 65 genera, were recovered using both methods. In total, 11 isolates are potentially new bacterial species, and 34 were associated with 22 species described recently. The iChip method yielded a higher diversity of microorganisms (47 genera) than the traditional method (38 genera) and was particularly effective in enriching Actinomycetota. Antibacterial screening against target bacteria showed that 85 isolates (22%) exhibited antibacterial activity. The Streptomyces, Pseudomonas, and Bacillaceae taxa accounted for most antibacterial-producing bacteria with some presenting promising undescribed characteristics. Other active isolates were affiliated with less-known antibacterial producers such as Arthrobacter, Chryseobacterium, Delftia, Ensifer, Flavobacterium, Rahnella, and Stenotrophomonas, among others. These results highlight the potential of our microbial collection as a source of new antibacterial natural products.

Development and validation of a one-step SMN assay for genetic testing in spinal muscular atrophy via MALDI-TOF MS.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disorder primarily attributed to the homozygous deletion of the survival motor neuron 1 (SMN1) gene, with disease severity closely correlated to the copy number variations (CNV) of SMN2. Conventional methodologies, however, fail to provide a comprehensive gene overview of SMN and are often both time-intensive and costly. In this study, we present a novel one-step MALDI-TOF MS assay for SMA gene testing. To accurately quantify CNV, we incorporated RPPH1 as an internal control alongside normal samples and competing templates targeting SMN1, SMN2, and RPPH1 for multiple corrections. The CNV assay enables precise quantification of exons 7/8 in both SMN1 and SMN2 genes, achieving a kappa value of 0.935 (P < 0.001) when compared with multiple ligation-dependent probe amplification (MLPA) during its development phase. This accuracy was further corroborated in a cohort comprising 78 individuals. To identify patients harboring compound heterozygous mutations or silent carriers, prevalent pathogenic variants along with sequence variants of SMN1 were integrated into our analysis framework; plasmids were constructed for methodological validation purposes. Utilizing these combinatorial assays for SMN detection, we identified one patient exhibiting a compound heterozygous mutation characterized by genotype [0 + 1d] and another subject presenting genotype [2 + 1], who harbored simultaneous variants of g.27134T > G and g.27706_27707delAT. The CNV assessment combined with pathogenic variants analysis developed through MALDI-TOF MS provides a comprehensive gene profile of SMN within a single analytical run. Given its unparalleled cost-effectiveness and time efficiency, this approach holds significant promise for further application in clinical diagnosis as well as newborn screening for SMA.

Automatic identification of clinically important Aspergillus species by artificial intelligence-based image recognition: proof-of-concept study.

While morphological examination is the most widely used for Aspergillus identification in clinical laboratories, PCR-sequencing and MALDI-TOF MS are emerging technologies in more financially-competent laboratories. However, mycological expertise, molecular biologists and/or expensive equipment are needed for these. Recently, artificial intelligence (AI), especially image recognition, is being increasingly employed in medicine for fast and automated disease diagnosis. We explored the potential utility of AI in identifying Aspergillus species. In this proof-of-concept study, using 2,813, 2,814 and 1,240 images from four clinically important Aspergillus species for training, validation and testing, respectively; the performances and accuracies of automatic Aspergillus identification using colonial images by three different convolutional neural networks were evaluated. Results demonstrated that ResNet-18 outperformed Inception-v3 and DenseNet-121 and is the best algorithm of choice because it made the fewest misidentifications (n = 8) and possessed the highest testing accuracy (99.35%). Images showing more unique morphological features were more accurately identified. AI-based image recognition using colonial images is a promising technology for Aspergillus identification. Given its short turn-around-time, minimal demand of expertise, low reagent/equipment costs and user-friendliness, it has the potential to serve as a routine laboratory diagnostic tool after the database is further expanded.

Porin expression in clinical isolates of Klebsiella pneumoniae: a comparison of SDS-PAGE and MALDI-TOF/MS and limitations of whole genome sequencing analysis

BackgroundThe permeability of the outer membrane barrier modulates the susceptibility of microorganisms to antimicrobial agents. Loss or structural alterations of porins contribute to decreased antibiotic concentration of multiple antimicrobial agents. Precise definition of porin profiles is of critical importance to understand the role of porins in antimicrobial resistance. The objectives of this study are to compare the expression patterns of major outer membrane proteins (OMP) of clinical isolates of Klebsiella pneumoniae obtained with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF/MS), with those obtained with sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and to correlate porin expression patterns with the sequences of porins genes defined with whole genome sequencing (WGS).MethodsThe OMP profiles of 26 clinical isolates of K. pneumoniae and of strain ATCC 13883 (wild-type) and ATCC 700603 (producing SHV-18) have been determined using both SDS-PAGE and MALDI-TOF/MS. SDS-PAGE was performed using both homemade and commercial gels, and protein bands were identified by liquid chromatography coupled to mass spectrometry. A rapid extraction method was used to analyse OMPs by MALDI-TOF/MS. The sequences of porin genes were obtained by WGS and mutations were defined by BLAST.ResultsSame results were obtained for all strains either using SDS-PAGE or MALDI-TOF/MS. SDS-PAGE showed protein bands of ~ 35, ~36, and ~ 37 kDa, identified as OmpA, OmpK36 and OmpK35, respectively. By MALDI-TOF/MS, peaks at ~ 35,700 (OmpA), ~ 37,000 (OmpK35), and ~ 38,000 (OmpK36) m/z were detected. ompK35 was intact in nine wild-type isolates and was truncated in 13 isolates, but OmpK35 was not observed in 3 isolates without mutations in ompK35. One point mutation was detected in another isolate and multiple mutations were detected in the remaining isolate. ompK36 was truncated in two isolates lacking this protein and presented one point mutation (n = 1) or multiple mutations in the remaining isolates.ConclusionMALDI-TOF/MS was reliable for porin detection, but because of the complex regulation of porin genes, WGS cannot always anticipate protein expression, as observed with SDS-PAGE and MALDI-TOF/MS.

Salivary peptidomic profiling of chronic gingivostomatitis in cats by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and nanoscale liquid chromatography-tandem mass spectrometry.

Chronic gingivostomatitis in cats (FCGS) is a moderately to severely painful condition, potentially caused by inadequate immune response to oral antigenic stimulation. Salivary peptidome analysis can identify inflammatory protein mediators and pathways involved in oral mucosal immune activation and may indicate potential therapeutic options for FCGS. Evaluate the diversity and abundance of salivary peptides in cats with FCGS using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Thirty-two cats with FCGS and 18 healthy controls. Case-control cross-sectional study. We compared the salivary peptide profiles of diseased and healthy cats. The diagnosis of FCGS was confirmed by histopathology. Saliva samples were analyzed for viral infections using polymerase chain reaction (PCR), peptide mass fingerprint (PMF) using MALDI-TOF MS, and peptide identification using nano LC-MS/MS. Distinct clusters of peptide profiles were observed between groups. In FCGS, 26 salivary peptides were altered, including apolipoprotein A1, nuclear receptor subfamily 1 group I member 3, fibrinogen alpha chain, interleukin 2 receptor gamma, interleukin 23 receptor, hemoglobin subunit alpha, and serpin peptidase inhibitor clade A (alpha-1 antiproteinase, antitrypsin) member 12, protein-tyrosine-phosphatase, and cholinergic receptor nicotinic alpha 10 subunit. Protein-anti-inflammatory drug interaction networks were observed. Peptide mass fingerprint and peptide profiles identified distinct clusters between FCGS and healthy cats. The 9 novel salivary peptide markers were associated with the JAK/STAT and PI3K/Akt pathways and immune responses. These potentially noninvasive biomarkers may facilitate understanding of FCGS pathophysiology and guide future therapeutic research.

Efficiency of MALDI-TOF MS at identifying and discriminating immature stages of cimex lectularius and cimex hemipterus bed bugs

Over the last two decades, an increase in bed bug infestations has been observed worldwide. Although their definitive role as vectors of infectious agents has not yet been demonstrated, bed bugs have a direct effect on human health through dermatological reactions to their bites and psychological disorders linked to domestic infestations. In this study, the effectiveness of using MALDI-TOF MS to correctly identify these two bed bug species at immature stages was assessed, as well as it effectiveness as discriminating between the immature stages (IS) of C. lectularius and C. hemipterus and their associated developmental stages. A total of 305 specimens were subjected to MALDI-TOF MS analysis, including 153 C. lectularius (28 eggs and 25 nymphs per stage from IS1 to IS5) and 152 C. hemipterus (27 eggs and 25 nymphs per stage from IS1 to IS5). ). MALDI-TOF MS analysis enabled us to obtain 84.97% (130/153) of high-quality MS spectra in terms of reproducibility and profile intensity. Twenty-four spectra including two per stage, from egg to IS5, and per bed bug species - were added to our in-house MS reference arthropod spectra database. All specimens were correctly identified at the species level, independently of the developmental stage, with log score values (LSVs) ranging from 1.75 to 2.79 (mean = 2.29 ± 0.12) and 1.81 to 2.71 (mean = 2.37 ± 0.03) for C. lectularius and C. hemipterus, respectively. MALDI-TOF MS correctly classified 53,33% (104/195) of the Cimex at the correct immature stage. Conversely, an accurate comparison of the profiles with a Genetic Algorithm model underlined that grouping the immature stages in two groups, early (IS1-IS2) and late (IS3-IS4-IS5), made it possible to obtain a cross validation (CV) and recognition capability (RC) greater than 92% and 94%, respectively, for both species. This study holds great promise for the management of bed bug infestations.

Outbreak of Esophagitis and Ingluvitis Caused by Salmonella Typhimurium in Passeriform Birds of the Genus Sporophila Seized from Wildlife Trafficking.

The occurrence of esophagitis and ingluvitis caused by Salmonella Typhimurium in passerines seized from illegal wildlife trafficking is described. This illegal activity causes stress and leads to lowered immunity in the birds. Additionally, inadequate hygiene conditions predispose the birds to diseases such as salmonellosis. Few studies report the occurrence of Salmonella-induced lesions in the esophagus and crop of passerines; therefore, this study aimed to describe the disease in birds of the genus Sporophila, as well as to investigate the presence of the bacterium in the environment and determine the antimicrobial resistance profile of the isolated bacteria. Three birds of the genus Sporophila were necropsied. In the esophagus and crop, yellowish plaques corresponding to necrosis with bacterial aggregates were observed. Salmonella spp. was isolated from these lesions, with genus confirmation via MALDI-TOF mass spectrometry. Environmental samples were collected from the enclosures and cages where the animals were quarantined, and the same bacterium was isolated. In two birds, the serotype S. Typhimurium was identified. Antibiograms performed on the strains from the birds and the environment revealed resistance to antibiotics in the classes of penicillins, sulfonamides, aminoglycosides, monobactams, tetracyclines, and first and third-generation cephalosporins. To the authors' knowledge, this is the first report of this agent causing death in Sporophila due to esophagitis and ingluvitis. It is also the first report of salmonellosis in three species of passerines in Brazil. The study underscores the importance of understanding the pathogens circulating in wild animals, especially within the context of One Health.

Survey in ruminants from Rwanda revealed high diversity and prevalence of extended-spectrum cephalosporin-resistant Enterobacterales

BackgroundAntimicrobial resistance (AMR) in Enterobacterales constitutes a significant threat to the health of both humans and animals and a socioeconomic problem. Enterobacterales, mainly Escherichia coli, carrying β-lactamases has become one of the main indicators to estimate the burden of AMR in animals within “One Health” approach.ObjectivesTo assess the presence of extended-spectrum cephalosporin-resistant Enterobacterales associated with ruminants (cattle, sheep, goats) habituated in all five provinces of Rwanda and to perform in depth characterization of isolates.MethodsWe screened 454 rectal swabs from 203 cows, 170 goats, and 81 sheep and selective isolation of extended-spectrum cephalosporin-resistant Enterobacterales was conducted. Isolates were identified as a members of the order Enterobacterales by MALDI-TOF MS and further characterized by susceptibility testing and by whole-genome sequencing.ResultsOut of the 454 samples, 64 extended-spectrum cephalosporin-resistant Enterobacterales were isolated from 58 animals. Isolates belonged to seven bacterial species and were identified as Escherichia coli (n = 54), Enterobacter bugandensis (n = 4), Enterobacter mori (n = 2), Klebsiella pneumoniae (n = 2), Enterobacter dykesii (n = 1), and Citrobacter freundii (n = 1). All isolates displayed an Extended-spectrum β-lactamases (ESBL) phenotype, with exception of Citrobacter freundii isolate displayed both an ESBL and AmpC phenotype. In addition, all Enterobacter isolates were identified as stably de-repressed AmpC-producers. ESBLs genes, blaCTX−M−15 was predominant. Resistance to tetracycline and tet(A) was most frequently observed among non-β-lactam resistance. Forty-eight isolates displayed multidrug-resistance phenotypes. A shiga toxin-producing E. coli and an enterotoxigenic E. coli isolate were observed. Genome comparisons revealed thirty-five E. coli sequence types (ST) (ST10, ST307 being predominate).ConclusionsConsidering the high proximity between ruminants and humans in Rwanda, the dissemination of antimicrobial drug resistance highlights the public health threats and requires the joint and multisectoral action of human and veterinary medicine, at human-animal-environment interfaces. Therefore, it is important to establish national and global “One Health” surveillance programs of AMR to tackle the antibiotic-resistant crisis in human and veterinary medicine.

Reduction of Staphylococcus aureus biofilms by lactic acid bacteria isolated from fermented transitional milk

ABSTRACT This study aimed to evaluate the inhibitory effect of lactic acid bacteria (LAB) isolated from fermented transition milk against Staphylococcus aureus from cows with mastitis adhered to teat cup rubber and stainless steel surfaces of cows. Transition milk, collected from multiparous Holstein cows, was fermented for 33 days. LAB were isolated based on their different colony morphologies on MRS agar and identified using MALDI-TOF MS analysis. The antagonistic activity was determined by the double-layer diffusion assay and the inhibition of S. aureus biofilms by LAB supernatant. Among the LAB isolates evaluated, Lacticaseibacillus paracasei, Lacticaseibacillus casei, Lactobacillus rhamnosus, Enterococcus faecalis, and Enterococcus faecium were identified. Out of the fifteen LAB isolates, nine exhibited zones of inhibition ranging from 12.53 to 25.83 mm against two strains of S. aureus. Lower concentrations of both S. aureus strains adhered to stainless steel and rubber coupons were detected after 20 min of contact with the supernatants of three selected LAB when compared to the control. These findings suggest that the metabolites produced by the selected LAB could potentially be used as an alternative method to inhibit and control antimicrobial-resistant S. aureus strains, as well as reduce their biofilm formation.

Nasal Carriage of Antimicrobial-Resistant Staphylococci by Fallow Deer (Dama dama) Taken in a Natural Park of Tuscany, Central Italy.

Wild animals are recognized as significant reservoirs for various zoonotic pathogens, including antibiotic-resistant bacteria. This study aimed to investigate the presence of Staphylococcus spp. strains in fallow deer (Dama dama) inhabiting a natural preserve in Central Italy and to examine the phenotypic and genotypic antimicrobial resistance and the presence of some virulence genes among the isolates. During July and December 2022, nasal swabs were collected from 175 fallow deer, which were then analyzed through bacteriological cultures. In total, 176 Staphylococcus spp. strains were isolated and subsequently identified using MALDI-TOF mass spectrometry. S. aureus was the most abundant species with 66 (37.5%) strains, followed by S. hyicus, 34 (19.31%) strains, S. sciuri, 32 (18.18%) strains, S. chromogenes, 27 (15.34%) strains, S. xylosus, 11 (6.25%) strains, S. warneri, 5 (2.84%) strains, and S. devriesei, 1 (0.56%) strain. Antimicrobial susceptibility was assessed for each isolate via the agar disk diffusion method, testing a panel of 13 molecules belonging to 9 antimicrobial classes. The highest resistance rates were detected for penicillin (29.55%), rifampicin (22.73%), and amikacin (20.45%). Notably, intermediate susceptibility was observed for erythromycin (61.93%), enrofloxacin (28.41%), and ceftiofur (21.02%). Conversely, the strains exhibited particularly high susceptibility to amoxicillin/clavulanic acid (99.43%), cefoxitin (97.73%), and vancomycin (96.02%). Based on the results, 32 (18.18%) isolates were classified as multidrug-resistant (MDR). Two strains of S. chromogenes and one strain of S. xylosus, both resistant to penicillin, tested positive for the blaZ gene. No methicillin-resistant strains were found, and none of the isolates harbored genes associated with enterotoxin and toxic shock syndrome toxin production. This study highlights the potential role of wildlife, particularly fallow deer, as reservoirs of antibiotic-resistant Staphylococcus spp. strains. Such findings underscore the importance of monitoring wildlife for antimicrobial resistance, which could have implications for public health and veterinary medicine.

An antimicrobial drug recommender system using MALDI-TOF MS and dual-branch neural networks.

Timely and effective use of antimicrobial drugs can improve patient outcomes, as well as help safeguard against resistance development. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently routinely used in clinical diagnostics for rapid species identification. Mining additional data from said spectra in the form of antimicrobial resistance (AMR) profiles is, therefore, highly promising. Such AMR profiles could serve as a drop-in solution for drastically improving treatment efficiency, effectiveness, and costs. This study endeavors to develop the first machine learning models capable of predicting AMR profiles for the whole repertoire of species and drugs encountered in clinical microbiology. The resulting models can be interpreted as drug recommender systems for infectious diseases. We find that our dual-branch method delivers considerably higher performance compared to previous approaches. In addition, experiments show that the models can be efficiently fine-tuned to data from other clinical laboratories. MALDI-TOF-based AMR recommender systems can, hence, greatly extend the value of MALDI-TOF MS for clinical diagnostics. All code supporting this study is distributed on PyPI and is packaged at https://github.com/gdewael/maldi-nn.

An antimicrobial drug recommender system using MALDI-TOF MS and dual-branch neural networks

Timely and effective use of antimicrobial drugs can improve patient outcomes, as well as help safeguard against resistance development. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently routinely used in clinical diagnostics for rapid species identification. Mining additional data from said spectra in the form of antimicrobial resistance (AMR) profiles is, therefore, highly promising. Such AMR profiles could serve as a drop-in solution for drastically improving treatment efficiency, effectiveness, and costs. This study endeavors to develop the first machine learning models capable of predicting AMR profiles for the whole repertoire of species and drugs encountered in clinical microbiology. The resulting models can be interpreted as drug recommender systems for infectious diseases. We find that our dual-branch method delivers considerably higher performance compared to previous approaches. In addition, experiments show that the models can be efficiently fine-tuned to data from other clinical laboratories. MALDI-TOF-based AMR recommender systems can, hence, greatly extend the value of MALDI-TOF MS for clinical diagnostics. All code supporting this study is distributed on PyPI and is packaged at https://github.com/gdewael/maldi-nn.

Isolation, Genomics-Based and Biochemical Characterization of Bacteriocinogenic Bacteria and Their Bacteriocins, Sourced from the Gastrointestinal Tract of Meat-Producing Pigs.

Antimicrobial resistance (AMR) poses a significant challenge to animal production due to the widespread use of antibiotics. Therefore, there is an urgent need for alternative antimicrobial strategies to effectively manage bacterial infections, protect animal health, and reduce reliance on antibiotics. This study evaluated the use of emerging approaches and procedures for the isolation, identification, and characterization of bacteriocin-producing bacteria and their bacteriocins, sourced from the gastrointestinal tract (GIT) of meat-producing pigs. Out of 2056 isolates screened against Gram-positive and Gram-negative indicator strains, 20 of the most active antimicrobial isolates were subjected to whole genome sequencing (WGS) for the prediction of coding DNA sequences (CDS) and the identification of bacteriocin gene clusters (BGC) and their functions. The use of an in vitro cell-free protein synthesis (IV-CFPS) protocol and the design of an IV-CFPS coupled to a split-intein mediated ligation (IV-CFPS/SIML) procedure made possible the evaluation of the production and antimicrobial activity of described and putatively novel bacteriocins. A colony MALDI-TOF MS procedure assisted in the identification of class I, II, and III lanthipeptides. MALDI-TOF MS and a targeted proteomics, combined with a massive peptide analysis (LC-MS/MS) approach, has proven valuable for the identification and biochemical characterization of previously described and novel bacteriocins encoded by the isolated bacteriocin-producing strains.

Abstract A032: Performance evaluation of a new protein stabilizing blood collection tube with a novel whole blood separation device for clinical plasma proteomics

Abstract Introduction: We have developed a novel blood separation device (BCD) which separates plasma from cellular components by lateral flow and stabilizes the sample for transport to a testing laboratory. Patient blood is drawn into an anticoagulant blood collection tube (BCT) and then applied to the separation device. There is outstanding interest in the field of clinical proteomics regarding the formulation in BCTs and whether they would also additionally the plasma proteome. This is especially critical where hemoglobin may provide interference to the analyte being measured. We evaluated the performance of a newly available plasma protein stabilizing BCT in combination with the BCD and other currently available K2EDTA BCT (no stabilization). Methods: Whole blood from normal healthy donors was drawn into protein stabilizing BCT and a comparator anticoagulant BCT. At baseline, 24-, 48-, 72- and 120-hour time points, a 250 μL aliquot of whole blood from each tube was spotted on to the BCD to allow separation of the cellular components and plasma. All of the test specimens on the spotted devices were allowed to dry overnight to replicate time in transit. After drying, proteins were subjected to MALDI-TOF MS (mass spectrometry) after extraction with water and co- crystallization with sinapinic acid. Spectra were acquired in triplicate and m/z feature intensities for hemoglobin were assessed in R. Results: Visual inspection of the new protein stabilizing and K2EDTA BCTs showed the steady accumulation of hemoglobin over time in the plasma layer in the latter tubes. After spotting on the BCD and MALDI-TOF analysis, plasma from the protein- stabilizing BCT showed no significant increases in either the hemoglobin α or β subunits over the five (5) day period. This was consistent across all donors (heme α p = 0.7933, heme β p = 0.6425) and at all timepoints assessed. In the K2EDTA BCT, both the α and β hemoglobin subunits increased significantly over time and across all donors (heme α p &amp;lt; 0.0001, heme β p = 0.0232). The hemoglobin α subunit accumulated in the plasma component to a greater degree than did the β subunit (heme α slope = 4.863E-5, heme β slope = 1.398E-5). These data suggest that the formulation in the protein stabilizing BCT may attenuate red blood cell hemolysis to a greater degree as compared to conventional K2EDTA blood collection tubes. Additionally, the mean reproducibility for both hemoglobin subunits was lower when the protein stabilizing BCT was used than when the anticoagulant BCT was used across all donors at all time points (protein stabilizing BCT x ̅ CV = 8.5%, anticoagulant BCT x ̅ CV = 8.9%). Conclusions: The use of the newly available protein stabilizing BCT with the BCD reduces the accumulation of hemoglobin in the plasma component while improving reproducibility when compared to the currently used anticoagulant BCT. Interference by heme was significantly attenuated as measured by a sensitive MALDI-ToF assay. Citation Format: Colin McDowell, Nylev Vargas, Gary A Pestano. Performance evaluation of a new protein stabilizing blood collection tube with a novel whole blood separation device for clinical plasma proteomics [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A032.

Catalytic Syntheses of Thiol-End-Functionalized ROMP Polymers.

Thiol-functionalized polymers have become a crucial class of materials due to their distinct chemical properties and versatile reactivity, leading to a broad spectrum of applications. Herein, we report the straightforward syntheses of a wide range of thiol-end-functionalized ring-opening metathesis polymerization (ROMP) polymers exploiting our previously reported catalytic ROMP mechanisms using suitable chain transfer agents. All the synthesized polymers were characterized via SEC, 1H NMR spectroscopy and MALDI-ToF mass spectrometry techniques. Furthermore, the existence of thiol groups on the polymer chains was verified through the well-established thiol coating reaction on gold nanoparticle surfaces. We believe this method of synthesizing thiol-end-functionalized ROMP polymers (using a reduced amount of ruthenium metal compared to conventional living ROMP) will be of great importance to materials science and biochemical research.

Investigation of Carriers of Salmonella and Other Hydrogen Sulphide-Positive Bacteria in the Digestive Content of Fish from the Atlantic Area of Macaronesia: A Comparative Study of Identification by API Gallery and MALDI-TOF MS.

Salmonella spp. are known pathogens in fish, with their presence potentially resulting from the contamination of the aquatic environment or improper handling. Accurate bacterial identification is crucial across various fields, including medicine, microbiology, and the food industry, and thus a range of techniques are available for this purpose. In this study, Salmonella spp. and other hydrogen sulphide-positive bacteria were investigated in the digestive contents of fish destined for consumption from the Atlantic area of Macaronesia. Two identification techniques were compared: the traditional API method and the MALDI-TOF MS technique. For the identification of Salmonella spp. carriers, 59 samples were processed following ISO 6579-1:2017. A total of 47 strains of Gram-negative bacilli were obtained. No Salmonella spp. isolates were detected. The most frequent genus was Enterobacter (76.50%), followed by Shewanella (10.63%). The MALDI-TOF MS technique showed a high concordance with the API technique, with 72.34% concordance at the species level. Both techniques demonstrated a high degree of concordance in the identification of Enterobacter cloacae, with 87.23% genus-level concordance and 12.76% non-concordant identifications. This study highlights the limitations of the API technique and the speed and precision of MALDI-TOF MS. The identified bacteria could pose a health risk to humans.