Matrix-assisted Laser Desorption Ionization-time Research Articles

Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment-Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel Congeria jalzici, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches.

Rhodococcus infection: a 10-year retrospective analysis of clinical experience and antimicrobial susceptibility profile.

Rhodococcus equi is an opportunistic pathogen known to cause pulmonary and extrapulmonary disease among immunocompromised patients. Treatment is frequently challenging due to intrinsic resistance to multiple antibiotic classes. While non-equi Rhodococcus spp. are prevalent, their clinical significance is poorly defined. There is also limited data on antibiotic susceptibility testing (AST) of Rhodococcus infection in humans. We conducted a single-center, retrospective cohort study evaluating clinical characteristics, microbiologic profile, and AST of Rhodococcus infections between June 2012 and 2022 at our tertiary academic medical center. Identification of Rhodococcus spp. was performed by Sanger 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization-time of flight mass spectrometry, and AST was performed by agar dilution. Three hundred twenty-two isolates of Rhodococcus spp. were identified from blood (50%), pulmonary (26%), and bone/joint (12%) sources. R. equi/hoagii, R. corynebacterioides, and R. erythropolis were the most frequently isolated species, with 19% of isolates identified only to genus level. One hundred ninety-nine isolates evaluated for AST demonstrated high-level resistance to amoxicillin/clavulanate, cephalosporins, and aminoglycosides. More than 95% susceptibility to imipenem, vancomycin, linezolid, rifampin, and clarithromycin was observed. Non-equi species showed a significantly more favorable AST profile relative to R. equi. Clinically significant Rhodococcus infection was rare with 10 cases diagnosed (majority due to R. equi) and managed. The majority of patients received 2- or 3-drug combination therapy for 2-6 months, with favorable clinical response. Significant differences in AST were observed between R. equi and non-equi species. Despite high antimicrobial resistance to several antibiotic classes, imipenem and vancomycin remain appropriate empiric treatment options for R. equi. Future research evaluating mechanisms underlying antimicrobial resistance is warranted.

The Role of Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass Spectrometry (MALDI-TOF-MS) in the Identification of Non-fermenting Gram-Negative Bacilli from Clinical Isolates in a Tertiary Care Hospital

Non-fermenting Gram-negative bacilli (NFGNB) are a heterogeneous group of aerobic non-spore forming bacilli. Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass Spectrometry (MALDI-TOF-MS) is a quick, precise diagnostic method that could identify the isolates at the species level. The aim of the study is to identify the non-fermenters isolated from various clinical infections by MALDI-TOF analysis and to detect the antibiogram of these isolates. The non-fermenters were isolated from various clinical specimens, plated on Blood agar and MacConkey agar and incubated at 37⁰C for 24 hours. Identification was done on the basis of colony morphology, Gram stain, and MALDI-TOF Analysis. Antimicrobial Susceptibility Testing was done by Kirby- Bauer Disc Diffusion Technique using commercially available antibiotics on Muller-Hinton Agar. A total of 360 samples were studied. The majority of the non-fermenters were from Blood/ Body fluids (39.72%). Acinetobacter spp (31.67%) was the most common isolate, followed by Pseudomonas spp (22.50%). Other non-fermenters like Stenotrophomonas maltophilia, Burkholderia spp, Elizabethkingia spp, Achromobacter spp, Chryseobacterium spp, Ralstonia spp, Pandoraea spp, Rhizobium radiobacter, Sphingomonas spp, Ochrobactrum spp, Myroides odoratimimus and Brevundimonas spp were rarely isolated non-fermenters. Most of the strains were multi drug resistant to commonly used antibiotics like Cephalosporins, Aminoglycosides and Carbapenems. Our results showed that identifying non-fermenters using MALDI-TOF MS is a reliable and rapid technique that shortens the time to initiate appropriate therapy and even the length of the hospital stay.

Association of ITGA4 and ICAM-1 gene polymorphisms with the risk and clinicopathological characteristics of Crohn's disease

To assess the association between the polymorphism of integral protein α4 (ITGA4) and intercellular adhesion molecule 1 (ICAM-1) genes and the risk and clinicopathological characteristics of Crohn's disease (CD) among Chinese patients. From January 2010 to January 2021, a total of 215 CD patients and 529 gender- and age-matched healthy controls were enrolled from the Second Affiliated Hospital of Wenzhou Medical University as the study subjects. Genotypes of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) were determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Harvey-Bradshaw Index (HBI) was applied to assess the disease activity of CD, and the patients were further divided into subgroups based on the Montreal Classification Criteria of CD. Unconditional logistic regression was employed to analyze the distribution of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) polymorphisms between the patients and healthy controls and their association with the clinicopathological characteristics of the patients. The frequencies of T allele and CT+TT genotypes of ITGA4 (rs7562325) were higher in CD patients than the healthy controls (40.70% vs. 31.57%, P = 0.001; 62.79% vs. 54.36%, P = 0.042). The G variant and AG+GG genotypes of ITGA4 (rs6740847) were less common in patients with moderately to severely active CD compared with those with mildly active CD (31.18% vs. 51.72%, P = 0.002; 55.91% vs. 75.86%, P = 0.042). However, the opposite conclusion was drawn for the G allele (G) and AG+GG genotypes of ICAM-1 (rs5498) (31.45% vs. 17.24%, P = 0.027; 54.30% vs. 31.04%, P = 0.020). Compared with patients with terminal ileal or ileocolic CD, G allele and AG+GG genotypes of ITGA4 (rs6740847) were more prevalent in patients with colonic CD (55.26% vs. 29.38%, P < 0.001; 84.21% vs. 53.11%, P<0.001). The same conclusion could also be drawn for the G allele and AG+GG genotypes of ICAM-1 (rs5498) (42.11% vs. 26.84%, P = 0.008; 73.69% vs. 46.33%, P = 0.002). The frequency of homozygous GG genotype of ICAM-1 (rs5498) was lower in patients with stricturing and penetrating CD than those with non-stricturing and non-penetrating CD (0.00% vs. 12.32%, P = 0.001). The G allele and AG+GG genotypes of the ITGA4 (rs6740847) were more common in patients with perianal lesions than those without (40.28% vs. 30.77%, P = 0.049; 72.22% vs. 51.75%, P = 0.004). Variants of the ITGA4 (rs7562325) may be a risk factor for CD, whilst those of the ITGA4 (rs6740847) may be associated with the decline of disease activity and risk for colon involvement and perianal lesions. Variants of the ICAM-1 (rs5498) may increase the risk of disease activity and colonic involvement in CD patients, however, it may be a protective factor for stenosis and penetration. In addition, variants of the ITGA4 (rs6740847) and ICAM-1 (rs5498) may be associated with the early onset of CD.

Enterobacter cloacae from urinary tract infections: frequency, protein analysis, and antimicrobial resistance

The genus Enterobacter belongs to the ESKAPE group, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. This group is characterized by the development of resistance to various antibiotics. In recent years, Enterobacter cloacae (E. cloacae) has emerged as a clinically important pathogen responsible for a wide range of healthcare-associated illnesses. Identifying Enterobacter species can be challenging due to their similar phenotypic characteristics. The emergence of multidrug-resistant E. cloacae is also a significant problem in healthcare settings. Therefore, our study aimed to identify and differentiate E. cloacae using Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a fast and precise proteomic analytical technique. We also tested hospital-acquired E. cloacae isolates that produce Extended-spectrum beta-lactamases (ESBL) against commonly used antibiotics for treating urinary tract infections (UTIs). We used a total of 189 E. cloacae isolates from 2300 urine samples of patients with UTIs in our investigation. We employed culturing techniques, as well as the BD Phoenix™ automated identification system (Becton, Dickinson) and Analytical Profile Index (API) system for the biochemical identification of E. cloacae isolates. We used the MALDI Biotyper (MBT) device for peptide mass fingerprinting analysis of all isolates. We utilized the single peak intensities and Principal Component Analysis (PCA) created by MBT Compass software to discriminate and cluster the E. cloacae isolates. Additionally, we evaluated the sensitivity and resistance of ESBL-E. cloacae isolates using the Kirby Bauer method. Out of the 189 E. cloacae isolates, the BD Phoenix system correctly identified 180 (95.24%) isolates, while the API system correctly identified 165 (87.30%) isolates. However, the MBT accurately identified 185 (98.95%) isolates with a score of 2.00 or higher. PCA positively discriminated the identified E. cloacae isolates into one group, and prominent peaks were noticed between 4230 mass-to-charge ratio (m/z) and 8500 m/z. The ESBL-E. cloacae isolates exhibited a higher degree of resistance to ampicillin, amoxicillin-clavulanate, cephalothin, cefuroxime, and cefoxitin. Several isolates were susceptible to carbapenems (meropenem, imipenem, and ertapenem); however, potential future resistance against carbapenems should be taken into consideration. In conclusion, MALDI-TOF MS is a powerful and precise technology that can be routinely used to recognize and differentiate various pathogens in clinical samples. Additionally, the growing antimicrobial resistance of this bacterium may pose a significant risk to human health.

Identification of environmental Actinobacteria in buildings by means of chemotaxonomy, 16S rRNA sequencing, and MALDI-TOF MS.

The manuscript addresses the challenges in identifying environmental bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Biotyper-based protein profiling. The matter of the studies-actinobacterial strains-has been isolated mostly from building materials that originated from a confirmed moisture-damaged situation. Polyphasic taxonomy, 16S RNA gene sequencing, and MALDI-TOF mass spectrometry were applied for identification purposes. In this experimental paper, a few important facts are highlighted. First, Actinobacteria are abundant in the natural as well as built environment, and their identification on the species and genus levels is difficult and time-consuming. Second, MALDI-TOF MS is an effective tool for identifying bacterial environmental strains, and in parallel, continuous enrichment of the proteomics mass spectral databases is necessary for proper identification. Third, the chemical approach aids in the taxonomical inquiry of Actinobacteria environmental strains.

Molecular characterization of carbapenem-resistance in Gram-negative isolates obtained from clinical samples at Jimma Medical Center, Ethiopia.

In resource-constrained settings, limited antibiotic options make treating carbapenem-resistant bacterial infections difficult for healthcare providers. This study aimed to assess carbapenemase expression in Gram-negative bacteria isolated from clinical samples in Jimma, Ethiopia. A cross-sectional study was conducted to assess carbapenemase expression in Gram-negative bacteria isolated from patients attending Jimma Medical Center. Totally, 846 Gram-negative bacteria were isolated and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Phenotypic antibiotic resistance patterns were determined using the Kirby-Bauer disk diffusion method and Etest strips. Extended-spectrum β-lactamase phenotype was determined using MAST disks, and carbapenemases were characterized using multiplex polymerase chain reactions (PCR). Among the isolates, 19% (157/846) showed phenotypic resistance to carbapenem antibiotics. PCR analysis revealed that at least one carbapenemase gene was detected in 69% (107/155) of these strains. The most frequently detected acquired genes were blaNDM in 35% (37/107), blaVIM in 24% (26/107), and blaKPC42 in 13% (14/107) of the isolates. Coexistence of two or more acquired genes was observed in 31% (33/107) of the isolates. The most common coexisting acquired genes were blaNDM + blaOXA-23, detected in 24% (8/33) of these isolates. No carbapenemase-encoding genes could be detected in 31% (48/155) of carbapenem-resistant isolates, with P. aeruginosa accounting for 85% (41/48) thereof. This study revealed high and incremental rates of carbapenem-resistant bacteria in clinical samples with various carbapenemase-encoding genes. This imposes a severe challenge to effective patient care in the context of already limited treatment options against Gram-negative bacterial infections in resource-constrained settings.

A New Case of Fournier's Gangrene Caused by Actinotignum schaalii

Actinotignum schaalii (formerly known as Actinobaculum schaalii) is an anaerobic or facultative anaerobic gram-positive bacillus that can be found commensally in the urogenital region. It can be overlooked because it grows slowly and is difficult to identify with classical microbiology laboratory techniques. Colonies become visible after 48-72 hours of incubation on blood agar in anaerobic or CO₂-rich media. While it typically causes urinary tract infection in older individuals, cases of bacteremia, vertebral osteomyelitis, endocarditis and cellulitis have been reported. Fournier's gangrene caused by A.schaalii has been reported very rarely so far. Fournier's gangrene has been defined as necrotizing fasciitis of the external genitalia, perineal and perianal region. Diabetes, immunosuppression, peripheral vascular disease, urethral anomalies, chronic alcoholism and smoking are important predisposing factors. In addition, approximately 25% of the cases have no known or identifiable etiology. The bacteria causing the infection may originate from skin, urogenital or intestinal microbiota. In this case report, a new case of Fournier's gangrene caused by A.schaalii was presented. A 65-year-old male patient admitted to the emergency department with the complaints of pain, swelling, redness in the left testis and also nausea, vomiting and chills that started three days ago. Physical examination revealed increased diameter of the scrotum, intense hyperemia of the skin and foci of necrosis. It was learned that the patient had no known chronic disease other than benign prostatic hyperplasia. The patient reported smoking of 25 packs of cigarettes per year. Routine laboratory tests revealed leukocyte= 32.41 x 109/L, neutrophil= 89.9%, procalcitonin= 1.62 ug/L, CRP= 265.07 mg/L and the patient was operated with the diagnosis of Fournier's gangrene. Gram staining of the abscess specimen obtained during the operation showed gram-positive bacilli both inside and outside the leukocytes. After 24 hours, grampositive bacilli were detected in the Gram staining of thin, transparent/gray colonies grown on 5% sheep blood and chocolate agar. The isolate was identified as A.schaalii by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) VITEK® MS (bioMérieux, France) microbial identification system. VITEK®2 ID ANC (bioMérieux, France) bacterial identification card was also used for comparison but the bacteria could be identified. As a result of the sequence analysis performed for confirmation, it was shown to be 100% homologous with Actinobaculum schaalii (GenBank accession no: FJ711193.1). For susceptibility tests, 5% sheep blood Schaedler agar was used and incubated in anaerobic environment. According to the minimal inhibitory concentration (MIC) results evaluated after 48 hours, penicillin was found to be 0.032 mg/L, clindamycin 0.125 mg/L, ciprofloxacin 0.19 mg/L, ceftazidime 4 mg/L, and amoxicillin 0.19 mg/L. The primary cause that initiated the infection in the case could not be identified, but it was thought that the presence of prostatic hyperplasia and smoking history may have contributed to the occurence or the progress of the disease. It is noteworthy that the ciprofloxacin MIC result was quite low compared to other studies. In addition, this study revealed the value of MALDI-TOF MS based methods in identification. In conclusion, it is thought that a significant proportion of A.schaalii infections may be overlooked due to the difficulty in growth and identification. Increasing the diagnostic power of clinical microbiology laboratories for poorly identified bacteria and renewing the databases of commercial identification systems are important for the early and accurate diagnosis and treatment of serious infections that may occur with such agents.

Retraction for Belkacemi et al., "Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity".

Retraction for Belkacemi et al., "Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity".

Clinico-epidemiological Analysis of Stenotrophomonas Maltophilia Bacteraemia, its Associated Risk Factors and Outcome in Patients Admitted at a Tertiary Care Centre in Northern India- A Retrospective Cohort Study

Introduction: Stenotrophomonas maltophilia is a Gram-negative, motile, and glucose non fermenting bacterium commonly found in hospital settings. It poses a significant risk to immunocompromised individuals, often causing nosocomial infections. Aim: To identify the risk factors associated with Stenotrophomonas maltophilia bacteraemia and compare the factors influencing patient survival and mortality. Materials and Methods: Clinical and laboratory data from 39 cases of Stenotrophomonas bacteraemia encountered between July 2021 and July 2022 in the Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India, were analysed in the present retrospective study in August 2022. The study included all cultures positive for S. maltophilia bacteraemia, identified through Matrix-assisted Laser Desorption Ionisation Time- Of-Flight Mass Spectrometry (MALDI-TOF-MS). Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method, following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Statistical analysis and outcome assessment were conducted using Statistical Package for Social Sciences (SPSS) version 20.0. Results: Clinical data from all 39 bacteraemia patients were extracted from the hospital information system for analysis. The mean age of the patients included in the present study was 46.0±20.29 years, with a male predominance of 27 (69.23%). The most common risk factors associated with S. maltophilia bacteraemia were the presence of an indwelling catheter in 21/39 cases (53.8%) and co-existing pulmonary infections in 18/39 cases (46.2%). S. maltophilia isolates exhibited high susceptibility to Minocycline (94.87%), Ticarcillin-Clavulanic acid (87.18%), Levofloxacin (84.62%), and Cotrimoxazole (84.62%). The 30-day mortality rate was reported as 28.20% (11/39). Conclusion: S. maltophilia can cause various infections in immunocompromised patients. The appropriate use of empirical antibiotics and strict adherence to infection control measures can reduce hospital stays, as well as 14-day and 30- day mortality rates among affected patients.

Antifungal Susceptibility Testing and Cluster Analysis of Candida auris Strains.

Candida auris is an opportunistic pathogen that has become widespread in recent years and shows resistance to multiple drugs. The aim of our study was to determine the antifungal susceptibilities of C. auris isolates, to perform a dendrogram from the mass spectra of strains obtained from matrix-assisted laser desorption ionization-time of flight mass spectrometry in order to evaluate the proteomic similarities of the strains and determine the geographical clade of C. auris strains in this study by the help of Multiplex RT-PCR. The samples yielded 58 C. auris isolates. MALDI TOF MS (BioMerieux, France) was used for identification of the isolates and Sensititre Yeast One (Thermoscientific) system was used for antifungal susceptibility testing. Dendrograms of strain's spectra were generated by using the RUO/Saramis (BioMerieux, France) database and evaluated through hierarchical clustering analysis. The selected nine strains were examined at the clade level by using Multiplex RT-PCR. Results The susceptibility profile of the strains revealed resistance to Fluconazole in 84% (MICs ≥ 32) and resistance to Amphotericin B in 60% (MIC ≥ 2). All strains were found to be sensitive to Anidulafungin and Micafungin. The dendrogram of the main spectra of C. auris isolates showed a similarity range of 35 - 100%. The nine strains studied were identified as clade 1 (South Asian). It was determined that C. auris strains were members of geographical clade 1, and the Amphotericin B resistance was found to be higher than expected. This situation poses a threat to critically ill patients.

Phenotypic and genotypic detection of Beta-lactamase producing Pseudomonas aeruginosa isolated from Haryana, India

Antimicrobial resistance causes substantial risks to human health globally, and millions of people die worldwide due to multiple drug resistances. Beta-lactam drugs are common for curing infections, and resistance to these drugs cause serious threat to humans. The resistance is acquired by the gram-negative Pseudomonas aeruginosa by producing beta-lactamases such as metallo beta-lactamase (MBL), extended-spectrum beta-lactamase enzymes (ESBL), and AmpC β-lactamases. Hence, this study was intended to detect the occurrence of MBL, ESBL, and AmpC β-lactamases producing P. aeruginosa and to evaluate antibiotic sensitivity at the Pandit Bhagwat Dayal Sharma, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India. A total of 163 P. aeruginosa were isolated from the different samples of patients, such as urine, blood, sputum, pus, and pleural fluids. The P. aeruginosa was characterized morphologically, biochemically, and with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Their antibiotic sensitivity was evaluated by the Kirby-Baur disc diffusion method. Antibiotic sensitivity tests of P. aeruginosa showed 163/163 were susceptible to Polymyxin-B, 78/163 and 65/163 were resistant against Ceftazidime (CAZ) and IMP antibiotics, respectively. The IMP, CAZ, and cefoxitin-resistant isolates were selected and further evaluated for ESBL, MBL, and AmpC enzyme production. In conclusion, the findings of this study indicated a significant presence of ESBL, MBL, and AmpC enzyme-producing P. aeruginosa among the patients. The ESBL prevalence was much higher in indoor patients than in outdoor patients. The total prevalence of MBL-producing strains in Imipenem-resistant P. aeruginosa (IRPA) was (46/62) 74.19%, which is an alarming signal. There was a higher prevalence of IRPA MBL-producing strains in indoor patients (36/46) 78.6% as compared to outdoor patients (10/16) 62.50%. Identification of bronchoalveolar lavage and sputum was also done using the Biofire Film Array, which revealed the resistant genes, including NDM (20 genes), CTX-M (17 genes), OXA-48-like (9 genes), VIM (5 genes), and IMP (2 genes). Antibiotics like cefotaxime and CAZ have less effect, but carbapenems and aminoglycosides are the best options for treating ESBL-producing P. aeruginosa. Drugs not recommended for treating this pathogen are penicillins and sulfonamides like co-trimoxazoles. Strict infection control measures, careful monitoring of antibiotic administration, and routine screening for ESBL-producing strains are advised before treating the patients.

Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.

Identification of Autoantigens in Pediatric Gastric Juices.

This study aimed to investigate the presence of autoantigens in the gastric juices of children. Gastric juice and serum samples were obtained from 53 children <15 years of age who underwent gastric endoscopy. Among these, 8, 22, and 23 participants were in the age groups 0-5, 6-10, and 11-15 years, respectively. These samples were analyzed using two-dimensional electrophoresis (2-DE), immunoblot analysis, and matrix-assisted laser desorption ionization-time of-flight mass spectrometry. Furthermore, we reviewed the histopathological findings and urease test results and compared them with the results of 2-DE and immunoblot analysis. There were no statistically significant differences in urease test positivity, grades of chronic gastritis, active gastritis, or Helicobacter pylori infiltration of the antrum and body among the three age groups. Three distinct patterns of gastric juice were observed on 2-DE. Pattern I was the most common, and pattern III was not observed below the age of 5 years. Histopathological findings were significantly different among active gastritis (p=0.037) and H. pylori infiltration (p=0.060) in the gastric body. The immunoblots showed large spots at an approximate pH of 3-4 and molecular weights of 31-45 kDa. These distinct, large positive spots were identified as gastric lipase and pepsin A and C. Three enzymes, which are normally secreted under acidic conditions were identified as autoantigens. Further investigation of the pathophysiology and function of autoantigens in the stomach is required.

Differential Protein Expression between the Motor and Sensory Fascicles in Rat Femoral Nerve Injury.

It is important to distinguish between motor and sensory fascicles of the peripheral nerves for nerve alignment in surgery. However, there are no biomarkers currently available for effective identification of motor or sensory fascicles. The objective of this study was to identify differentially expressed proteins between motor and sensory fascicles of rats in response to injury. The study was carried out using a rat femoral nerve injury model. A proteomic analysis was performed to detect differential protein expression using samples of bilateral motor and sensory branches of intact and injured rat femoral nerves through fluorescent two-dimensional difference gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Chi-square tests and t-tests were performed for comparison between motor or sensory nerve groups. The data identified six proteins that were differentially expressed between motor and sensory fascicles (>1.5-fold, P < 0.05), including apolipoprotein E, neurofilament light polypepticle, TEC kinase, serine protease inhibitor A3N, peroxiredoxin-2, and TPM1. The proteomic results were consistent with the mRNA expression levels of these genes as determined by quantitative reverse transcription polymerase chain reaction. Our study suggests that these proteins may play roles in nerve regeneration and repair. Importantly, apolipoprotein E and Serpina3n may serve as specific biomarkers for distinguishing motor and sensory fascicles of the peripheral nerves for nerve alignment in surgery.

Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

An Outbreak of Ralstonia mannitolilytica Septicaemia at a Tertiary Care Hospital: An Observational Cross-sectional Study

Introduction: Ralstonia spp. is an emerging non fermenting Gram negative bacillus implicated in cases of bloodstream infections in immunocompromised individuals. It is commonly found as an environmental contaminant in hospital settings. Several sporadic outbreaks have been reported from different parts of the world due to Ralstonia spp. This study reports a similar outbreak at a tertiary care hospital in Northern India. Aim: To determine the source of Ralstonia septicaemia in affected patients at a tertiary care centre. Materials and Methods: The present observational crosssectional study was conducted at the Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, Uttar Pradesh, India from February 2020 until the end of March 2020 (two months). A total of 2,650 blood cultures were received during the study period; of these, 53 (2%) patients were found to have Ralstonia mannitolilytica infection over a two month period. All patients from various wards whose blood cultures showed growth of Ralstonia species were included in this study. The organism was identified using both biochemical tests and Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS). Antibiotic sensitivity testing was conducted using the Kirby-Bauer disk diffusion assay. Environmental surveillance was conducted to detect the source of origin. Patients’ age, sex, duration of hospital stay, co-morbidities, and other clinical parameters were recorded. Statistical analysis was performed using Microsoft Excel. Results: There were 52 cases of septicaemia due to Ralstonia mannitolilytica, and one Ralstonia isolate was obtained from intraoperative pus. Most of the Ralstonia isolates obtained were Multidrug-Resistant (MDR), showing resistance to imipenem, meropenem, amikacin, aztreonam, and sensitivity to first-line drugs such as ceftazidime, piperacillin-tazobactam, cefoperazone-sulbactam, levofloxacin, and trimethoprimsulfamethoxazole, resulting in successful treatment. Out of 53 cases, one patient succumbed to death due to surgical complications. Environmental sampling did not yield any organisms resembling Ralstonia spp. Conclusion: The environmental source of the Ralstonia bacteraemia outbreak could not be identified in this study. All patients except one were successfully treated with antibiotics. Clinicians and microbiologists should remain vigilant in case any such case arises to prevent further outbreaks.

Enhanced exopolysaccharide production in gamma irradiated Bacillus subtilis: A biofilm-mediated strategy for ZnO nanoparticles removal

Biofilm-mediated strategy was studied for ZnO nanoparticle removal from aqueous media. Bacillus subtilis isolated from the soil rhizosphere was selected based on its high viscosity (133 Pa/s) of the cultivated culture and biofilm formation. The bacterium was exposed to gamma-irradiation to enhance EPS production along with its cultivation in EPS-producing media. The results show an increase in viscosity that reached 160 Pa/s at 2 kGy. EPS production increased from 4.45 to 7.95 mg/mL and the protein/carbohydrate ratio increased from 3 to 4.4 which reflects the stickiness of EPS. Thermal Gravimetric Analysis (TGA) showed 2 phase weight loss for gamma irradiated EPS and defined protein peaks when characterized using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). Native and gamma-irradiated Bacillus subtilis cells with their enhanced EPS were grown as a biofilm on sterile waste gauze fabric, Scanning Electron Microscopy (SEM) showed an increased biofilm attachment in gamma-irradiated samples. The latter was used for the removal of ZnO NP from aqueous media. Energy dispersive X-ray (EDX) mapping confirms that ZnO NPs were entrapped within the carbon and oxygen elements forming the biofilm with net intensities of 14.04, 1713, and 1190, respectively. The results confirm that biofilm-mediated strategy is effective in nanoparticles removal.

Trihexyphenidyl Alters Its Host's Metabolism, Neurobehavioral Patterns, and Gut Microbiome Feedback Loop-The Modulating Role of Anacyclus pyrethrum.

Trihexyphenidyl (THP)-a synthetic anticholinergic medication used to manage parkinsonism and extrapyramidal symptoms-has gained significant clinical recognition. However, there is a critical gap in understanding its withdrawal effects. This study investigates the intricate interplay between gut microbiota and oxidative stress during THP withdrawal. Furthermore, it explores the therapeutic potential of Anacyclus pyrethrum (AEAP) for alleviating the associated adverse effects. This comprehensive research combines behavioral tests, biochemical analysis, gut microbiome assessment utilizing matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and oxidative stress measures. The results reveal that the chronic administration of THP leads to severe withdrawal syndrome, marked by heightened anxiety, depressive-like behaviors, increased cortisol levels, elevated oxidative stress, and gut dysbiosis. However, the administration of AEAP alongside THP shows a significant capacity to mitigate these deleterious effects. Co-treatment and post-treatment with AEAP increased bacterial density and diversity, promoting the proliferation of beneficial bacteria associated with improved gut health. Furthermore, AEAP administration reduced cortisol levels and exhibited potent antioxidant properties, effectively countering the THP-induced oxidative damage. This study highlights the withdrawal effects of THP and underscores the therapeutic potential of AEAP for managing these symptoms. The findings reveal its promising effects in alleviating behavioral and biochemical impairments, reducing oxidative stress, and restoring gut microbiota, which could significantly impact the clinical management of THP withdrawal and potentially extend to other substance withdrawal scenarios.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, β-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.