Use of the proteomic tool MALDI-TOF MS in termite identification

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) has proved effective for the identification of many arthropods. A total of 432 termite specimens were collected in Mali, Cote d’Ivoire, Togo, Senegal, Switzerland and France. Morphologically, 22 species were identified, including Ancistrotermes cavithorax, Amitermes evuncifer, Cryptotermes brevis, Cubitermes orthognathus, Kalotermes flavicollis, Macrotermes bellicosus, Macrotermes herus, Macrotermes ivorensis, Macrotermes subhyalinus, Microcerotermes parvus, Microtermes sp., Odontotermes latericius, Procubitermes sjostedti, Promirotermes holmgreni, Reticulitermes grassei, Reticulitermes lucifugus, Reticulitermes santonensis, Trinervitermes geminatus, Trinervitermes occidentalis, Trinervitermes togoensis, Trinervitermes sp., Trinervitermes trinervoides and Trinervitermes trinervius. Analysis of MALDI-TOF MS spectra profiles from termites revealed that all were of high quality, with intra-species reproducibility and inter-species specificity. Blind testing of the spectra of 389 termites against our updated database with the spectra of 43 specimens of different termite species revealed that all were correctly identified with log score values (LSVs) ranging from 1.65 to 2.851, mean 2.290 ± 0.225, median 2.299, and 98.4% (383) had LSVs > 1.8. This study is the first on the use of MALDI-TOF for termite identification and shows its importance as a tool for arthropod taxonomy and reinforces the idea that MALDI-TOF MS is a promising tool in the field of entomology.

MALDI-TOF MS Based Bacterial Antibiotics Resistance Finger Print for Diabetic Pedopathy

Diabetes mellitus has become a major global health issue. Currently, the use of antibiotics remains the best foundational strategy in the control of diabetic foot infections. However, the lack of accurate identification of pathogens and the empirical use of antibiotics at early stages of infection represents a non-targeted treatment approach with a poor curative effect that may increase the of bacterial drug resistance. Therefore, the timely identification of drug resistant bacteria is the key to increasing the efficacy of treatments for diabetic foot infections. The traditional identification method is based on bacterial morphology, cell physiology, and biochemistry. Despite the simplicity and low costs associated with this method, it is time-consuming and has limited clinical value, which delays early diagnosis and treatment. In the recent years, MALDI-TOF MS has emerged as a promising new technology in the field of clinical microbial identification. In this study, we developed a strategy for the identification of drug resistance in the diagnosis of diabetic foot infections using a combination of macro-proteomics and MALDI MS analysis. The macro-proteomics result was utilized to determine the differential proteins in the resistance group and the corresponding peptide fragments were used as the finger print in a MALDI MS analysis. This strategy was successfully used in the research of drug resistance in patients with diabetic foot infections and achieved several biomarkers that could be used as a finger print for 4 different drugs, including ceftazidime, piperacillin, levofloxacin, and tetracycline. This method can quickly confirm the drug resistance of clinical diabetic foot infections, which can help aid in the early treatment of patients.

A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.

Antimicrobial susceptibility of commensal Neisseria in a general population and men who have sex with men in Belgium

Non-pathogenic Neisseria are a reservoir of antimicrobial resistance genes for pathogenic Neisseria meningitidis and Neisseria gonorrhoeae. Men who have sex with men (MSM) are at risk of co-colonization with resistant non-pathogenic and pathogenic Neisseria. We assessed if the antimicrobial susceptibility of non-pathogenic Neisseria among MSM differs from a general population and if antimicrobial exposure impacts susceptibility. We recruited 96 participants at our center in Belgium: 32 employees, 32 MSM who did not use antibiotics in the previous 6 months, and 32 MSM who did. Oropharyngeal Neisseria were cultured and identified with MALDI-TOF–MS. Minimum inhibitory concentrations for azithromycin, ceftriaxone and ciprofloxacin were determined using E-tests® and compared between groups with non-parametric tests. Non-pathogenic Neisseria from employees as well as MSM were remarkably resistant. Those from MSM were significantly less susceptible than employees to azithromycin and ciprofloxacin (p < 0.0001, p < 0.001), but not ceftriaxone (p = 0.3). Susceptibility did not differ significantly according to recent antimicrobial exposure in MSM. Surveilling antimicrobial susceptibility of non-pathogenic Neisseria may be a sensitive way to assess impact of antimicrobial exposure in a population. The high levels of antimicrobial resistance in this survey indicate that novel resistance determinants may be readily available for future transfer from non-pathogenic to pathogenic Neisseria.

Exploratory Study on Application of MALDI-TOF-MS to Detect SARS-CoV-2 Infection in Human Saliva

SARS-CoV-2 has caused a large outbreak since its emergence in December 2019. COVID-19 diagnosis became a priority so as to isolate and treat infected individuals in order to break the contamination chain. Currently, the reference test for COVID-19 diagnosis is the molecular detection (RT-qPCR) of the virus from nasopharyngeal swab (NPS) samples. Although this sensitive and specific test remains the gold standard, it has several limitations, such as the invasive collection method, the relative high cost and the duration of the test. Moreover, the material shortage to perform tests due to the discrepancy between the high demand for tests and the production capacities puts additional constraints on RT-qPCR. Here, we propose a PCR-free method for diagnosing SARS-CoV-2 based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling and machine learning (ML) models from salivary samples. Kinetic saliva samples were collected at enrollment and ten and thirty days later (D0, D10 and D30), to assess the classification performance of the ML models compared to the molecular tests performed on NPS specimens. Spectra were generated using an optimized protocol of saliva collection and successive quality control steps were developed to ensure the reliability of spectra. A total of 360 averaged spectra were included in the study. At D0, the comparison of MS spectra from SARS-CoV-2 positive patients (n = 105) with healthy healthcare controls (n = 51) revealed nine peaks that significantly distinguished the two groups. Among the five ML models tested, support vector machine with linear kernel (SVM-LK) provided the best performance on the training dataset (accuracy = 85.2%, sensitivity = 85.1%, specificity = 85.3%, F1-Score = 85.1%). The application of the SVM-LK model on independent datasets confirmed its performances with 88.9% and 80.8% of correct classification for samples collected at D0 and D30, respectively. Conversely, at D10, the proportion of correct classification had fallen to 64.3%. The analysis of saliva samples by MALDI-TOF MS and ML appears as an interesting supplementary tool for COVID-19 diagnosis, despite the mitigated results obtained for convalescent patients (D10).

Mechanisms of Action of Zishen Yutai pills in Treating Premature Ovarian Failure Determined by Integrating UHPLC-Q-TOF-MS and Network Pharmacology Analysis

Background: Zishen Yutai (ZSYT) pill, a patent Chinese medicine, has been widely used in the treatment of infertility, abortion, and adjunctive treatment of in vitro fertilization (IVF) for decades. Recently, the results of clinical observations showed that premature ovarian failure (POF) patients exhibited improved expression of steroids and clinical symptoms associated with hormone disorders after treatment with ZSYT pills. However, the pharmacological mechanism of action of these pills remains unclear.Methods: The components of ZSYT found in blood circulation were identified via ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) technique in the serum of POF mice after oral administration of ZSYT pills. The potential targets of components were screened using Traditional Chinese Medicine Systems Pharmacology Database, Traditional Chinese Medicine Database@Taiwan, Drugbank Database, PubChem, HIT, Pharmapper, and SwissTargetPrediction. The target genes associated with POF were collected from Online Mendelian Inheritance in Man Database, PharmGkb, Genecards, therapeutic target database, and Genetic Association Database. The overlapping genes between the potential targets of ZSYT components and the target genes associated with POF were clarified via protein-protein interaction (PPI), pathway, and network analysis.Results: Nineteen components in ZSYT pills were detected in the serum of POF mice after oral administration. A total of 695 ZSYT-related targets was screened, and 344 POF-related targets were collected. From the results of ZSYT-POF PPI analysis, CYP19A1, AKR1C3, ESR1, AR, and SRD5A2 were identified as key targets via network analysis, indicating their core role in the treatment of POF with ZSYT pills. Moreover, the pathway enrichment results suggested that ZSYT pills treat POF primarily by regulating neuroactive ligand-receptor interaction, steroid hormone biosynthesis, and ovarian steroidogenesis.Conclusions: We demonstrated that regulation of neuroactive ligand-receptor interaction, steroid hormone biosynthesis, and ovarian steroidogenesis are very likely to be therapeutic mechanism of ZSYT pills in treating POF. Our study suggests that combining the analysis of ZSYT pills components in blood in vivo in the POF models and network pharmacology prediction may offer a tool to characterize the mechanism of ZSYT pills in the POF.

Co-Ingestion of Natal Plums (Carissa macrocarpa) and Marula Nuts (Sclerocarya birrea) in a Snack Bar and Its Effect on Phenolic Compounds and Bioactivities

This study investigated the effect of co-ingesting Natal plums (Carissa macrocarpa) and Marula nuts (Sclerocarya birrea) on the bioaccessibility and uptake of anthocyanins, antioxidant capacity, and the ability to inhibit α-glucosidase. A Natal plum–Marula nut bar was made by mixing the raw nuts and the fruit pulp in a ratio 1:1 (v/v). The cyanidin-3-O-sambubioside (Cy-3-Sa) and cyanidin-3-O-glucoside content (Cy-3-G) were quantified using the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). Inclusion of Natal plum in the Marula nut bar increased the Cy-3-Sa, Cy-3-G content, antioxidants capacity and α-glucosidase inhibition compared to ingesting Marula nut separately at the internal phase. Adding Natal plum to the Marula nut bar increased bioaccessibility of Cy-3-Sa, Cy-3-G, quercetin, coumaric acid, syringic acid and ferulic acid to 80.2% and 71.9%, 98.7%, 95.2%, 51.9% and 89.3%, respectively, compared to ingesting the Natal plum fruit or nut separately.