MALDI-TOF MS Technique Research Articles

Isolation, Selection, and Identification of Keratinolytic Bacteria for Green Management of Keratin Waste.

The volume of difficult-to-process keratin waste is increasing as a result of rising global meat production. If not properly managed, this waste can contribute to environmental pollution and pose a threat to human and animal welfare. An interesting and more sustainable alternative is therefore the bioconversion of keratin using microorganisms and their enzymes. This work aimed to isolate bacteria from soil samples and zoonotic keratins and to evaluate their enzymatic capacity to degrade α- and β-keratin wastes. A total of 113 bacterial strains were isolated from environmental samples and subjected to taxonomic identification using the MALDI-TOF MS technique and to a two-step screening for proteolytic and keratinolytic activity. The ability to degrade a β-rich keratin substrate was observed in almost all of the strains isolated from soil and horsehairs. In contrast, when an α-rich keratin substrate was used, the highest levels of hydrolysis were observed only for Ker39, Ker66, Ker85, Ker100, and Ker101. Strains with the highest biodegradation potential were identified using molecular biology methods. Phylogenetic analysis of 16S rDNA gene sequences allowed the assignment of selected keratinolytic microorganisms to the genera Exiguobacterium, Priestia, Curtobacterium, Stenotrophomonas, Bacillus, Kocuria, or Pseudomonas. The results of this study are a promising precursor for the development of new, more sustainable methods of managing keratin waste to produce high-value hydrolysates.

The usefulness of the MALDI-TOF MS technique in the determination of dairy samples' microbial composition: comparison of the new EXS 2600 system with MALDI Biotyper platform.

This study compared the EXS 2600 system with the MALDI Biotyper for identifying microorganisms in dairy samples. Of the 196 bacterial isolates from milk, whey, buttermilk, cream, and dairy wastewater, the species and genus consistent identification between two systems showed 74% and 99%, respectively. However, the level of species identification rate exhibited a difference, which was higher in Zybio than in Bruker-76.0% and 66.8%, respectively. Notably, the EXS 2600 system performed better with certain yeast species and H. alvei, while the Biotyper excelled with Pseudomonas bacteria. Unique microbial compositions were found in 85% of dairy samples, with whey and buttermilk having the highest diversity. This research highlights the EXS 2600's potential as a reliable dairy microbial identification tool and underscores the need for a more diverse and comprehensive spectral database, despite the database's focus on clinical applications (as announced).

Implications of Sample Preparation Methods on the MALDI-TOF MS Identification of Spore-Forming Bacillus Species from Food Samples: A Closer Look at Bacillus licheniformis, Peribacillus simplex, Lysinibacillus fusiformis, Bacillus flexus, and Bacillus marisflavi.

This research underscores the criticality of tailored culture conditions and incubation periods for effective and accurate identification of spore-forming bacteria: Bacillus licheniformis, Peribacillus simplex, Lysinibacillus fusiformis, Bacillus flexus, and Bacillus marisflav, isolated from food samples, utilizing the MALDI-TOF MS technique. All isolated strains were confirmed as Gram-positive bacteria from diverse genera through 16S rDNA gene sequencing. To enhance the accuracy of the identification process, the study employed an optimization strategy involving a varied incubation time (ranging from 1 to 48 h) and two distinct sample preparation approaches-direct transfer facilitated by formic acid and protein extraction via ethanol. It was observed that matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) could successfully identify approximately 47% of the samples following a 24 h incubation period. The study emphasizes the critical role of sample preparation methods in enabling precise bacterial identification. Our findings reveal the necessity of tailoring the incubation time for each sample, as the optimum period for accurate identification fluctuated between 1 and 12 h. Further demonstrating the interplay between incubation time and spore quantity, our study used the Schaeffer-Fulton staining method to show that the lowest spore counts were detected between 5 and 8 h of incubation. This provides evidence that spore formation impacts bacterial identification. Our research thus deepens the understanding of spore-forming bacteria identification using MALDI-TOF MS and illuminates the various factors affecting the dependability and accuracy of this technique. Future research may explore additional variables, such as the effect of varying culture media, to further augment identification accuracy and gain a holistic understanding of spore-forming bacterial behavior in food samples. By enhancing our knowledge, these findings can substantially contribute to improving food safety and quality assurance strategies by enabling the more accurate and efficient identification of spore-forming bacteria in the food industry, thereby elevating the standards of food safety.

Prevalence of aerobically growing opportunistic bacteria in the nasopharyngeal microbiota of pregnant women living in rural and urban areas

Abstract The upper respiratory tract, including the nasopharynx, is inhabited by many microbial species. Still, the nasopharyngeal microbiota (or microbiome) of pregnant women is not well understood and may be influenced by a number of factors, including place of residence. Many changes occur during pregnancy, and their association with the respiratory microbiota is important for the health of the mother and for the developing fetus. The aim of this study was to compare the colonisation of the nasopharyngeal cavity by culturable aerobic bacteria in the microbiota of women with a physiological pregnancy course according to rural or urban place of residence. Nasopharyngeal swabs were collected from 37 pregnant women (17 from rural and 20 from urban areas) to detect the colonisation of aerobically growing culturable bacteria. Isolates were identified using microbiological culture methods and the MALDI-TOF MS technique. Bacteria were colonisers of the nasopharynx cavity in all cases tested. Accordingly, 97.3% (36/37) of the pregnant women were colonised by Gram-positive bacteria, mainly coagulase-negative (CoNS) and -positive (CoPS) Staphylococcus spp. and Corynebacterium spp. in both the rural and urban groups. Gram-negative Klebsiella variicola, Proteus mirabilis and Pseudomonas congelans colonised only 17.6% (3/17) of rural women and Stenotrophomonas maltophilia only 5.0% (1/20) of the urban women. To summarise, all pregnant women were colonised with bacteria in the nasopharyngeal cavity. The majority from both rural and urban residences were colonised with Gram-positive bacteria only. Gram-negative bacteria were isolated mainly from samples in the rural group.

Prevalence of Culturable Bacteria and Yeasts in the Nasopharynx Microbiota during the Physiological Course of Pregnancy.

The aim of the study was to compare the prevalence of the nasopharyngeal carriage of culturable microorganisms in the microbiota of asymptomatic women with a physiological pregnancy (PW) and nonpregnant women (NPW). Nasopharyngeal swabs were collected from 53 PW and 30 NPW to detect bacterial and fungal colonization. Isolates were identified using the culture method and the MALDI-TOF MS technique. The nasopharyngeal microbiota (NPM) partially differed between PW and NPW. These differences in the frequency of nasopharyngeal colonization between the PW and NPW groups were not statistically significant (p > 0.05); all cases were colonized by bacteria and only two cases in the PW group were colonized by yeasts, namely, Rhodotorula spp. High levels of staphylococcal colonization, including predominantly coagulase-negative staphylococci and S. aureus in the nasopharyngeal sample, were present in both groups. The reduced number of Gram-negative rods colonized in the cases studied was seen in samples from the NPW group, particularly with Enterobacterales, and anaerobic Cutibacterium spp. were isolated only in the PW group (p < 0.05). Moreover, a higher carriage rate of Enterobacter aerogenes colonization was statistically significant (p < 0.05) and correlated with the NPW group. Pregnancy may disturb the composition of the NPM represented by commensals and opportunistic bacteria and promote yeast colonization as compared to nonpregnant women.

High diversity of yeast species and strains responsible for vulvovaginal candidiasis in South-East Gabon.

Candida albicans generally remains the principal pathogenic yeast responsible for vulvovaginal candidiasis (VVC), although with variable prevalence. In this study, we evaluated the evolution of the prevalence of the non-Candida albicans Candida (NCAC) species and investigated the genotypic diversity and the population genetic structure of the circulating C. albicans strains associated with VVC in the vicinity of Franceville (Gabon). A total of 110 independent isolates were identified using both MALDI-TOF MS and conventional techniques. The population genetic structure of the C. albicans strains was determined by multiple locus variable-number tandem repeat analysis using 4 microsatellite markers. The mean and median age of the patients was 31 years. Seven patients had a mixed infection. C. albicans accounted for 62% (n=68) of the total isolates. NCAC were dominated by C. glabrata, followed by P. kudriavzevii, C. parapsilosis, C. tropicalis, M. guilliermondii, and C. nivariensis. The cluster analysis revealed a high diversity, with a total of 50 different genotypes. The most represented genotype was shared by only four strains, while the vast majority (39 strains) had a unique MLVA pattern. Geographic clusters were not detected. The study provides information on species distribution and possible changing epidemiology while reporting for the first time C. nivariensis in VVC in Africa. This study is also the first to investigate the genotypic diversity of the circulating C. albicans strains associated with VVC in Central Africa. Such analyses would help understand the molecular epidemiology of C. albicans.

Drug resistance profile and detection of genes responsible for methicillin resistance in Staphylococcus aureus isolated from municipal waste

Currently, we are facing the ever-increasing phenomenon of bacteria being resistant to antibiotics. It is the consequence of excessive and incorrect use of drugs. The phenomenon is a global problem affecting bacteria both in their hospital population and in the natural environment. Municipal waste is an environment conducive to the development of microorganisms, therefore it contains various groups of bacteria, including drug-resistant staphylococci. The aim of the study was to identify species of bacteria, determine their antibiotic resistance, and assess the occurrence of genes responsible for methicillin resistance in Staphylococcus aureus isolated from mixed municipal waste. Strains were isolated by Koch’s serial dilution method with the use of microbiological media. Species were identified using the MALDI TOF-MS technique, whereas the drug resistance profile was determined by disk diffusion and molecular PCR methods. 250 isolates of S. aureus were collected. The highest resistance found was to cefoxitin, erythromycin and tetracycline. Among the bacteria collected, resistance to 1, 2, 3 or 4 antibiotics at the same time was the most common, with a maximum of 10. Additionally, 45 (18%) MDR (multidrug-resistant) isolates were detected. Methicillin resistance was found by the disk diffusion test in 60 (24%) strains, while the mecA gene was detected in as many as 180 (72%) isolates.

Characterization of the salivary microbiome before and after antibiotic therapy via separation technique

In the present research, the MALDI-TOF MS technique was applied as a tool to rapidly identify the salivary microbiome. In this fact, it has been monitored the changes occurred in molecular profiles under different antibiotic therapy. Significant changes in the composition of the salivary microbiota were noticed not only in relation to the non antibiotic (non-AT) and antibiotic treatment (AT) groups, but also to the used media, the antibiotic therapy and co-existed microbiota. Each antibiotic generates specific changes in molecular profiles. The highest number of bacterial species was isolated in the universal culture medium (72%) followed by the selective medium (48% and 38%). In the case of non-AT patients, the prevalence of Streptococcus salivarius (25%), Streptococcus vestibularis (19%), Streptococcus oralis (13%), and Staphylococcus aureus (6%) was identified while in the case of AT, Streptococcus salivarius (11%), Streptococcus parasanguinis (11%), Staphylococcus epidermidis (12%), Enterococcus faecalis (9%), Staphylococcus hominis (8%), and Candida albicans (6%) were identified. Notable to specified that the Candida albicans was noticed only in AT samples, indicating a negative impact on the antibiotic therapy.The accuracy of the MALDI-TOF MS technique was performed by the 16S rRNA gene sequencing analysis—as a reference method. Conclusively, such an approach highlighted in the present study can help in developing the methods enabling a faster diagnosis of disease changes at the cellular level before clinical changes occur. Once the MALDI tool allows for the distinguishing of the microbiota of non-AT and AT, it may enable to monitor the diseases treatment and develop a treatment regimen for individual patients in relation to each antibiotic.Key pointsThe salivary microbiota of antibiotic-treated patients was more bacteria varietyMALDI-TOF MS is a promising tool for recording of reproducible molecular profilesOur data can allow to monitor the treatment of bacterial diseases for patients

Comparing the Efficacy of MALDI-TOF MS and Sequencing-Based Identification Techniques (Sanger and NGS) to Monitor the Microbial Community of Irrigation Water.

In order to intensify and guarantee the agricultural productivity and thereby to be able to feed the world's rapidly growing population, irrigation has become very important. In parallel, the limited water resources lead to an increase in usage of poorly characterized sources of water, which is directly linked to a higher prevalence of foodborne diseases. Therefore, analyzing the microorganisms or even the complete microbiome of irrigation water used for food production can prevent the growing numbers of such cases. In this study, we compared the efficacy of MALDI-TOF Mass spectrometry (MALDI TOF MS) identification to 16S rRNA gene Sanger sequencing of waterborne microorganisms. Furthermore, we analyzed the whole microbial community of irrigation water using high-throughput 16S rRNA gene amplicon sequencing. The identification results of MALDI-TOF MS and 16S rRNA gene Sanger sequencing were almost identical at species level (66.7%; 64.3%). Based on the applied cultivation techniques, Acinetobacter spp., Enterobacter spp., Pseudomonas spp., and Brevundimonas spp. were the most abundant cultivable genera. In addition, the uncultivable part of the microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidota, Patescibacteria, and Verrucomicrobiota. Our findings indicate that MALDI-TOF MS offers a fast, reliable identification method and can act as an alternative to 16S rRNA gene Sanger sequencing of isolates. Moreover, the results suggest that MALDI-TOF MS paired with 16S rRNA gene amplicon sequencing have the potential to support the routine monitoring of the microbiological quality of irrigation water.

Optical and quantitative sensing capability of phenolphthalein derived Schiff base chromo‐fluorogenic sensor for Cu2+

Accurate tracking of Cu2+ is of major significance for several fields from human health to the environment and industry. Hence, well-organized, sensitive, and accurate methods for Cu2+ recognition in solutions are in special request. Herein, a novel phenolphthalein-based fluorogenic probe has been constructed by integrating a phenolphthalein-based building block with an amine-containing molecule. The probe was explored as a“switch-off” fluorogenicsensorfor Cu2+signaling in the presence of interfering cations in ionic concentrations and also displayed perfect selectivity/sensitivity toward Cu2+and faster equilibrium response time than any other reportedfluorogenic probes. As a sensing platform, the probe has a moderately experimental LOD of 84.0 nM Cu2+ in HEPES:CH3CN (v:v, 1:1, pH = 7). Binding affinity tests, MALDI–TOF MS techniques and theoretically DFT computations demonstrated that Cu2+formed coordinate bonds with phenolic hydroxyl and –CH = N groups of FPAB. The probe was successfully utilized for Cu2+ sensing in theFPABcoated silica plates and cotton swabs. The present study offers a fast, visible, low-cost and precise process for Cu2+ sensing.

Evaluation of the Tooth Surface after Irradiation with Diode Laser Applied for Removal of Dental Microorganisms from Teeth of Patients with Gingivitis, Using X-ray Photoelectron (XPS) and Optical Profilometry (OP).

Gingivitis is accompanied by microorganisms, including pathogens, which must be eliminated to speed up the treatment of inflammation. Laser irradiation may be one of the safe methods for reducing tissue contamination on the tooth surface. The aim of the study was the assessment of the tooth surface in patients with gingivitis after the use of a diode laser to eliminate microorganisms living there. In the first stage of the research, microorganisms were isolated (Candida albicans, C. guilliermondii, Escherichia coli, Haemophilus parainfluenzae, Klebsiella oxytoca, Neisseria subflava, Rothia dentocariosa, Rothia mucilaginosa, Streptococcus pneumoniae) from three patients with gingivitis, their identification confirmed using the MALDI-TOF MS technique (matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry). Then, the irradiation process with a diode laser was optimized to a wavelength of 810 nm ± 10 nm in five variants to reduce microorganisms on the tooth. The tooth surface was analyzed by X-ray photoelectron spectroscopy (XPS) and optical profilometry (OP) before and after irradiation. 103 to 106 CFU were detected on a 0.4 cm2 tooth area. Nine types of bacteria and two types of fungi dominated among the microorganisms. The laser at the most effective biocidal dose of 25 W/15.000 Hz/10 µs, average = 3.84 W, with three uses after 15 s, increased the reduction of fungi from 57.97% to 93.80%, and bacteria from 30.67% to 100%. This dose also caused a decrease in the degree of oxidation and in the effect of smoothing on the treated surfaces.

A new Fe3+-selective, sensitive, and dual-channel turn-on probe based on rhodamine carrying thiophenecarboxaldehyde: Smartphone application and imaging in living cells

A new dual-channel probe based on rhodamine B derivative (MSB) was successfully designed, synthesized, characterized by Nuclear Magnetic Resonance (NMR) Spectroscopy, Fourier Transform Infrared Spectrophotometer (FTIR), Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), X-ray Photoelectron Spectroscopy (XPS), and Single Crystal X-rayDiffraction, and the sensing abilities toward Fe3+ cation have been demonstrated and the probe was successfully utilized for fluorescence imaging of Fe3+ in living cells. The probe demonstrated quite fast, sensitive, and selective response to Fe3+ by causing an extreme enhancement in UV–vis and fluorescence spectroscopy techniques in the buffered aqueous media which makes MSB a dual-channel probe. While the color of MSB solution was initially light yellow, it turned pink in the presence of Fe3+, which provided highly selective naked-eye determination among several ions as alkaline, alkaline-earth, and transition metal ions. After that, the probe was easily applied to paper strips and real samples such as drinking waters and supplementary iron tablets for sensing Fe3+ in an aqueous solution. The detection limit (LOD) and the response time of the probe were determined as 4.85x10-9 M and 4 min, respectively, which are quite lower compared with other rhodamine based Fe3+ sensors in the literature. According to Job’s plot, 1H NMR titration, MALDI-TOF MS, XPS, and DFT study techniques, the complexation ratio between MSB and Fe3+ was found as 1:1. Moreover, the spectral response was reversible with alternately addition of Fe3+ or Na2EDTA to the MSB solution. In addition, fluorescence imaging in NIH/3T3 mouse fibroblast cells and studies in real samples with a quite high recovery rate exhibited that the probe is qualified for detection of Fe3+ ion with multiple practical usages.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the diagnosis of microorganisms.

Microbiology culture is the gold standard method for identifying microorganisms. This identificationprotocol takes several days to complete. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that can identify different microorganisms quickly and accurately. The objective of this work was to evaluate the use of MALDI-TOF MS in the routine of clinical laboratories to identify microorganisms and to identify their resistance to antimicrobials. This study evaluated the relevance of the MALDI-TOF MS technique for microbiological diagnosis through a literature review. The authors found that MALDI-TOF MS can identify bacteria, fungi, viruses and parasites, even in blood cultures, and also serves to assess antimicrobial resistance. Thus, MALDI-TOF MS can become an indispensable tool in laboratory diagnosis.

Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the discrimination of Lacticaseibacillus species

The closely related species, Lacticaseibacillus casei, L. paracasei, L. rhamnosus, L. chiayiensis, and L. zeae, are difficult to accurately discriminate by conventional identification methods. In this study, the bioTyper and in-house database of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was evaluated to discriminate five Lacticaseibacillus species. From the mass spectra of 130 isolates aligned with databases, 118 strains were correctly identified. On the other hand, databases could not accurately differentiate 12 isolates such as L. casei, L. rhamnosus and L. chiayiensis because the same colony was identified as two species with similar score. To overcome the database's limitations, the mass spectra were analyzed to discover species-specific protein peaks. The peaks at 6731 ± 1, 6849 ± 1, 7008 ± 1, 7376 ± 1, and 2593 ± 1 m/z were specifically found in the reference strains of L. casei, L. paracasei, L. rhamnosus, L. chiayiensis, and L. zeae, respectively. These peaks confirmed that the five peaks were consistently present in each species using 130 strains isolated from food samples. Our results demonstrate the high-resolution of MALDI-TOF MS technique for rapid and accurate classification of five species when used with databases coupled to specific peaks.

Immunomodulatory Properties of Polysaccharide-Rich Young Green Barley (Hordeum vulgare) Extract and Its Structural Characterization.

Young green barley (YGB) water extract has revealed a beneficial impact on natural killer (NK) cells’ ability to recognize and eliminate human colon cancer cells, without any side effects for normal colon epithelial cells. The direct anticancer effect of the tested compounds has been also shown. The mixture of oligosaccharides found in this extract was characterized by chemical analyses and via FT-IR spectroscopy and MALDI-TOF MS techniques. The YGB preparation contained 26.9% of proteins and 64.2% of sugars, mostly glucose (54.7%) and fructose (42.7%), with a small amount of mannose (2.6%) and galactose (less than 0.5%). Mass spectrometry analysis of YGB has shown that fructose oligomers contained from 3 to 19 sugar units. The number of fructans was estimated to be about 10.2% of the dry weight basis of YGB. The presented results suggest the beneficial effect of the consumption of preparations based on young barley on the human body, in the field of colon cancer prevention.

Suppression of Thiol-Dependent Antioxidant System and Stress Response in Methicillin-Resistant Staphylococcus aureus by Docosanol: Explication Through Proteome Investigation

The present study was aimed to investigate the effect of docosanol on the protein expression profile of methicillin-resistant Staphylococcus aureus (MRSA). Thus, two-dimensional gel electrophoresis coupled with MALDI-TOF MS technique was utilized to identify the differentially regulated proteins in the presence of docosanol. A total of 947 protein spots were identified from the intracellular proteome of both control and docosanol treated samples among which 40 spots were differentially regulated with a fold change greater than 1.0. Prominently, the thiol-dependent antioxidant system and stress response proteins are downregulated in MRSA, which are critical for survival during oxidative stress. In particular, docosanol downregulated the expression of Tpx, AhpC, BshC, BrxA, and YceI with a fold change of 1.4 (p = 0.02), 1.4 (p = 0.01), 1.6 (p = 0.002), 4.9 (p = 0.02), and 1.4 (p = 0.02), respectively. In addition, docosanol reduced the expression of proteins involved in purine metabolic pathways, biofilm growth cycle, and virulence factor production. Altogether, these findings suggest that docosanol could efficiently target the antioxidant pathway by reducing the expression of bacillithiol and stress-associated proteins.

Highly selective thiazole-appended “switch off” fluorogenic sensor with a detection level of nM for Cu2+: DFT calculations and practically applications

Several precedents demonstrate that fluorogenic sensors are promising candidates for the recognition and reasonable control of metal ions like Cu2+ in the environment because of real-time monitoring, abundant acquirable signals and its facile operation. Therefore, a new benzothiazole-based colorimetric and fluorogenic sensor, 4-(benzo[d]thiazol-2-yl)-2-(((4-(phenylamino)phenyl)imino)methyl)phenol (TAPOH), has been constructed as a sensing material for Cu2+ in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and acetonitrile (ACN) (v:v, 3:7, pH:7) media. TAPOH could specifically recognize and rapidly respond to Cu2+ during 0.5 min. The emission intensity of the TAPOH quenched with the adding of Cu2+ (0–20.0 eqv), showing a fairly low LOD of 2.23 nM. The reaction mechanism of the TAPOH-Cu2+ was fully characterized through MALDI–TOF MS techniques and then supported by density functional theory (DFT) computations. The binding stoichiometry for TAPOH-Cu2+ was confirmed as 2:1 and the possible mechanism for TAPOH-Cu2+ was suggested depended on the chelation-enhanced fluorescence quenching (CHEQ) of the heavy atom effect of Cu2+. Additionally, the TAPOH coated silica plates were successfully utilized for the sensing of Cu2+. All outcomes demonstrated that TAPOH could conveniently be employed for tracking Cu2+ in solutions.

Identification of Bacteria Associated with Post-Operative Wounds of Patients with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Approach.

The bacterial infection of post-operative wounds is a common health problem. Therefore, it is important to investigate fast and accurate methods of identifying bacteria in clinical samples. The aim of the study was to analyse the use of the MALDI-TOF MS technique to identify microorganism wounds that are difficult to heal. The most common bacteria are Escherichia coli, Staphylococcus spp., and Enterococcus spp. We also demonstrate the effect of culture conditions, such as the used growth medium (solid: Brain Heart Infusion Agar, Mueller Hilton Agar, Glucose Bromocresol Purple Agar, and Vancomycin Resistance Enterococci Agar Base and liquid: Tryptic Soy Broth and BACTEC Lytic/10 Anaerobic/F), the incubation time (4, 6, and 24h), and the method of the preparation of bacterial protein extracts (the standard method based on the Bruker guideline, the Sepsityper method) to identify factors and the quality of the obtained mass spectra. By comparing the protein profiles of bacteria from patients not treated with antibiotics to those treated with antibiotics based on the presence/absence of specific signals and using the UniProt platform, it was possible to predict the probable mechanism of the action of the antibiotic used and the mechanism of drug resistance.

MALDI-TOF Mass Spectrometry Technology as a Tool for the Rapid Diagnosis of Antimicrobial Resistance in Bacteria.

Species identification by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a routine diagnostic process for infectious diseases in current clinical settings. The rapid, low-cost, and simple to conduct methodology is expanding its application in clinical microbiology laboratories to diagnose the antimicrobial resistance (AMR) in microorganisms. Primarily, antimicrobial susceptibility testing is able to be carried out either by comparing the area under curve of MALDI spectra of bacteria grown in media with antimicrobial drugs or by identifying the shift peaks of bacteria grown in media including 13C isotope with antimicrobial drugs. Secondly, the antimicrobial resistance is able to be determined through identifying (i) the antimicrobial-resistant clonal groups based on the fingerprints of the clone, (ii) the shift peak of the modified antimicrobial drug, which is inactivated by the resistance determinant, (iii) the shift peak of the modified antimicrobial target, (iv) the peak specific for the antimicrobial determinant, and (v) the biomarkers that are coproduced proteins with AMR determinants. This review aims to present the current usage of the MALDI-TOF MS technique for diagnosing antimicrobial resistance in bacteria, varied approaches for AMR diagnostics using the methodology, and the future applications of the methods for the accurate and rapid identification of AMR in infection-causing bacterial pathogens.

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry

Purpose: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Materials and Methods: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. Results: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. Conclusions: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.