MALDI-TOF MS Research Articles

Enrichment and MALDI-TOF MS Analysis of Phosphoinositides in Brain Tissue.

Triazolium cyclodextrin click cluster (+CCC) is an ideal scaffold to specifically bind phosphoinositides (PIPs) via multivalent electrostatic interaction. A new enrichment material, triazolium cyclodextrin click cluster-magnetic agarose bead conjugate (+CCC-MAB), was synthesized and applied to the PIP enrichment of brain tissue. The enriched sample was analyzed using MALDI-TOF MS in negative ion mode without any derivatization. The PIP extract of brain tissue is known to contain abundant lipid interferences. By employing magnetic pull-down separation using +CCC-MAB, we effectively removed the weak-binding interferences in the PIP extract, thereby improving the signal-to-noise ratio (S/N) of the PIPs. Our +CCC-MAB-based PIP enrichment enabled us to analyze 16 PIP species in brain tissue. Six species with high S/N were assigned by MS/MS, while the remaining 10 species with low S/N were characterized by an empirical selection guide based on the biological relevance of PIPs. We conclude that +CCC-MAB-based PIP enrichment is a promising MALDI sample preparation method for specific PIP analysis in brain tissue.

Burkholderia cepacia: Understanding Pathogenicity, Virulence Factors, and Therapeutic Strategies

Burkholderia cepacia, formerly described as Pseudomonas cepacia, belongs to the Burkholderia cepacia complex (Bcc), a collection of closely related gram-negative bacteria that is considered a leading organism in causing pulmonary infections in humans. In healthcare settings, Burkholderia species have the ability to colonise medical equipment and surfaces, posing a threat of nosocomial infections. Moreover, they exhibit inherently resistance to various classes of antibiotics, thus being nominated as multi-drug resistant pathogens (MDR), that is why treatment of infections caused by these organisms is challenging. Enzymes such as metalloproteases, serine proteases, metalloproteases, and other extracellular lipases play a major role in its pathogenesis. In addition to this, different components, such as pili, flagella, and lipopolysaccharide (LPS), have a role in both the motility of the bacterial cell as well as its adherence to host cells. Identification is usually done with different biochemical reactions such as late oxidase activity, oxidation of glucose, variable lysine decarboxylation, maltose, mannitol, and lactose, and hydrogen sulphide gas production. Advanced techniques like semi-nested PCR, real-time PCR, or MALDI-TOF mass spectrometry enable quick and precise identification.
 Key words: Multi-drug resistant, Polymerase chain reaction, Burkholderia cepacia complex, matrix assisted laser desorption ionisation, time of flight mass spectrometry

New type paraben decorated mono- and dispirocyclotriphosphazene compounds: Synthesis, characterization and DNA binding studies

Parabens are a group of chemical preservatives that are widely used in the cosmetics, pharmaceutical, and food industries. Although some studies have raised concerns about potential health risks associated with parabens, it is important to note that the presence of parabens in tumor tissue does not provide conclusive evidence that parabens cause cancer. This is an area that requires further research. For this purpose, six new mono- and dispirocyclotriphosphazene compounds decorated with paraben were synthesized and designed for the first time. Their structures were identified by MALDI-TOF MS, 1H, 31P NMR and FT-IR spectroscopy. Moreover, the molecular structure of Compound 8 was determined by X-Ray crystallography. The DNA interaction properties of the targeted molecules were investigated via agarose gel electrophoresis.

The Detection of Crown Gall Disease in Kahramanmaraş and Surrounding Provinces

Aims: The aim of the study is to isolate the bacterial agent causing crown gall disease in Kahramanmaraş and its surrounding provinces, determine its prevalence in the region, and conduct its identification. 
 Material and methods: Between June and September of 2017-2019, strains obtained from the shoots and galls of vines showing symptoms of galls in vineyards in Kahramanmaraş, Adıyaman, Kilis, Gaziantep, and Şanlıurfa provinces were subjected to pathogenicity tests, classical, molecular, and MALDI-TOF MS identification.
 Results and Discussion: In this study, the prevalence of crown gall disease in 46 vineyards showing disease symptoms was determined as follows: Kahramanmaraş 76.47%, Adıyaman 75%, Gaziantep 50%, Kilis 33.33%, and Şanlıurfa 20%. From the 46 contaminated vineyards in the study area, 24 pathogenic of R. vitis strains were obtained. In the pathogenicity test conducted with R. vitis strains on the Hatun Parmağı grape variety under greenhouse conditions, the most virulent strains were determined based on the weights of the galls formed, and they were identified as CU52-3/2, CU39-11/5, and CU39-8/1. According to classical tests, 13 strains showed 100% similarity to R. vitis, while molecular characterization with R. vitis-specific primers revealed that 17 strains had the virA gene, 13 strains had the pehA gene, 10 strsains had the virF gene, and 6 strains had the octopine-type plasmid. Ten strains, representing the sample, were identicated as R. vitis through MALDI-TOF MS analysis. This study revealed the impact of pathogenic strains with different virulences on the same grape variety. In line with this, of control methods against highly virulent bacterial agents in reducing losses in plants can be investigated for their applicability in field and orchard areas in subsequent studies.
 Conclusions: Research on different grape varieties is essential to develop varieties that are resistant or tolerant to R. vitis, the pathogen causing this prevalent disease in Turkish viticulture.

Characterization by MALDI-TOF MS and 16S rRNA Gene Sequencing of Aerobic Endospore-Forming Bacteria Isolated from Pharmaceutical Facility in Rio de Janeiro, Brazil

Bacillus and related genera are among the most important contaminants in the pharmaceutical production environment, and the identification of these microorganisms at the species level assists in the investigation of sources of contamination and in preventive and corrective decision making. The aim of this study was to evaluate three methodologies for the characterization of endospore-forming aerobic bacterial strains isolated from a pharmaceutical unit in Rio de Janeiro, Brazil. MALDI-TOF MS was performed using MALDI Biotyper® and VITEK® MS RUO systems, and complete 16S rRNA gene sequencing was performed using the Sanger methodology. The results showed the prevalence of the genera Bacillus (n = 9; 36.0%), Priestia (n = 5; 20.0%), and Paenibacillus (n = 4; 16.0%). Three (20.0%) strains showed <98.7% of DNA sequencing similarity on the EzBioCloud Database, indicating possible new species. In addition, the reclassification of Bacillus pseudoflexus to the genus Priestia as Priestia pseudoflexus sp. nov. is proposed. In conclusion, 16S rRNA and MALDI TOF/MS were not sufficient to identify all strains at the species level, and complementary analyses were necessary.

MOLECULAR DETECTION OF FUSOBACTERIUM NUCLEATUM IN IN KAZAKHSTANY PATIENTS WITH COLORECTAL CANCER

Colorectal cancer is the leading cause of cancer death worldwide. Fusobacterium nucleatum is thought to play an important role in the development of colorectal cancer. F. nucleatum there are many studies that prove that has a great influence on the oncogenesis of colorectal cancer, but the participation of the bacterium in this process remains unclear. The goal of our study is to find out in colorectal cancer tissue F. nucleatum was the definition. Biomaterials from 43 colorectal cancer patients from the National scientific cancer center were analyzed. To detect bacteria, DNA was extracted from the biomaterial and culture inoculation was carried out in parallel. F. nucleatum detection was performed by two methods using Matrix activated laser microbiological desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and quantitative real-time PCR detection. As a result, MALDI determined 14.58%, and qPCR detected 46.51% of bacteria. Thus, F. nucleatum can be a biomarker of colorectal cancer risk.

Insights into population adaptation and biodiversity of lactic acid bacteria in challenged date palm leaves silaging, using MALDI–TOF MS

Insights into population adaptation and biodiversity of lactic acid bacteria in challenged date palm leaves silaging, using MALDI–TOF MS

Microbiological Distribution, Antimicrobial Susceptibility and Risk Factors of Polymicrobial Infections in Diabetic Foot.

Diabetic foot infection (DFI) leads to poor prognosis and polymicrobial infections are usually the main cause. The study is to explore the microbiological distribution, antimicrobial drug susceptibility, and risk factors of polymicrobial infections in hospitalized patients with DFI. This retrospective study included 160 patients with DFI in Wagner's grades 2, 3, and 4. Deep necrotic tissue was used to acquire specimens for microbiological culture. VITEK-2 system and MALDI-TOF mass spectrometry were used to identify the bacterial isolates. The Kirby Bauer method was used for drug susceptibility tests. A total of 202 pathogens were isolated. The proportion of gram-negative bacilli (GNB, 62.4%, 126 of 202) was higher than that of gram-positive cocci (GPC, 37.6%, 76 of 202). The most prevalent GPC was Staphylococcus aureus in every Wagner grade, while the most common GNB varied in different Wagner grades. Linezolid was the most effective antibiotic for GPC in different Wagner grades. Imipenem was the most effective antibiotic for GNB in Wagner grade 2. Amikacin was the most effective antibiotic for GNB in Wagner grades 3 and 4. Polymicrobial infections existed only in Wagner grades 3 and 4 and increased the risk of amputation (p < 0.01). History of antibiotics, duration of diabetic foot, CRP, and lower extremity arterial disease were the independent risk factors of polymicrobial infections (p < 0.05). Clinicians should adjust the antibiotic as needed based on the results of drug susceptibility and clinical treatment effect among different Wagner grades. Particular attention should be given to the treatment of polymicrobial infections.

Preparation of polyoxometalate modified magnetic metal–organic framework and application to the enrichment of phosphopeptides

Herein, polyoxometalate modified magnetic metal–organic framework (Fe3O4@PDA@MOF-POM) was successfully prepared and then applied to the enrichment of phosphopeptides. Fe3O4@PDA@MOF-POM can bind with phosphopeptides through ZrO clusters provided by the MOF layer and metal oxide originated from Zr-W-POM. The magnetic nanomaterial had high sensitivity (0.1 fmol), good reusability (10 cycles), and high selectivity (α-casein: β-casein: bovine serum albumin = 1: 1: 5000). Coupling with MALDI-TOF MS determination, non-fat milk was selected to preliminarily explore the applicability of Fe3O4@PDA@MOF-POM. Furthermore, 241 phosphopeptides derived from 232 phosphoproteins and 99 phosphopeptides attributed to 89 phosphoproteins were identified in human serum and saliva by nano LC-MS. This work paves a new way for the recognition of phosphopeptides in bio-samples.

Combing through museum collections. A “museomic” application of ZooMS

AbstractThis article presents the application of Zooarchaeology by Mass Spectrometry (ZooMS) to osseous Longobard artifacts from the collection of the “Musei Reali di Torino” (MRT; Torino, Italy). Like most archaeological items made of worked bone/antler in museum collections, the raw material of such specimens is usually attributed to deer, often without accurate taxonomic attribution. Therefore, the main aim of the present investigation was to shed light on taxonomical aspects using biomolecular approaches.We first examined the collagen preservation of the artifacts, then we compared three sampling methods (invasive, eraser‐based, and bag‐based), and we evaluated the quality of the collagen fingerprint obtained. Overall, we found a good, albeit not optimal, biomolecular preservation status, even in heavily restored objects coming from the 19th‐century collections.Out of 37 specimens analyzed through matrix‐assisted laser desorption ionization–time of flight–mass spectrometry (MALDI‐TOF‐MS) and nanoHPLC‐tandem MS, 31 yielded usable data. The results confirmed the widespread use of cervid as the osseous raw material for comb‐making in Longobard times in Piedmont, but we also found that bovine bones (Bos but also other taxa belonging to family Bovidae, such as caprines) and equid bones were exploited—demonstrating opportunistic use of animal resources. As far as the method is concerned, the ZooMS peptide markers useful to distinguish between bovids and cervids (m/z 1580 vsm/z 1550) (Buckley et al., 2009) are more frequently detected when analyzing bone chips, that is, with the invasive sampling method, rather than collagen extracts obtained using non‐invasive techniques. Nonetheless, the eraser method (eZooMS) seems to be a good trade‐off between invasivity and quality of the information obtained: eZooMS sampling does not leave visible marks on the object and therefore can contribute to facilitating the routine application of biomolecular methods in the daily practice of museum conservation laboratories. Indeed, an important outcome of the present study has been the establishment of a close collaboration between museum and biomolecular specialists.Taken together, our results suggest that the Longobards had a preference towards locally available resources, although this work did not highlight a clear association between raw material (deer, cattle, and other bovids) and object typology (in the case of combs) or function, except for buttons. The overall information obtained by this study confirms the potential of biomolecular approaches for reconstructing the biography of museum objects with a long and complex life and demonstrates the value of zooarchaeological study of museum collections.

Preanalytical, Analytical and Postanalytical Analyses on Corynebacterium spp. and Actinomycetaceae in Urine Samples of Patients with Suspected Urinary Tract Infection-A Hypothesis-Forming Observational Study.

A hypothesis-forming exploratory cross-sectional assessment was conducted to assess the occurrence and relevance of Gram-positive rod-shaped bacteria like Corynebacterium spp. and Actinomycetaceae in human urine samples. In total, 1170 urine samples from 1031 inpatients with suspected urinary tract infection were assessed for culture-based growth of Gram-positive rod-shaped bacteria applying API Coryne assays, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and in-house 16S rRNA gene sequencing. Overall, 502 different bacterial colonies from 346 urine samples taken from 324 inpatients were observed. The three quantitatively most abundant genera or genus clusters were Corynebacterium (254 isolates, 62%), Actinomyces/Winkia (79 isolates, 19%), and Actinotignum/Actinobaculum (29 isolates, 7%). Compared to sequencing, the diagnostic accuracy of all assessed competitor assays from the diagnostic routine was <80% for differentiation on the genus level and <30% for differentiation on the species level. Prolongated incubation for 4 days compared to 2 days resulted in additional detection of 15% of the totally recorded Gram-positive rod-shaped bacteria. An approximately 5-fold increased detection rate in mid-stream urine compared to urine acquired applying alternative sampling strategies was observed. In conclusion, in the rare event of the suspected clinical relevance of such findings, confirmatory testing with invasively sampled urine should be considered due to the high contamination rate observed in mid-stream urine. Confirmatory testing by DNA-sequencing methods should be considered if an exact identification of genus or species is regarded as relevant for the individual choice of the therapeutic strategy.

Changes in inoculated Salmonella enterica subsp. enterica Serovar Enteritidis and other microbiological qualities of vacuum-packed carrot slices after treatment with aqueous extract of Lobularia maritima

After harvesting, pathogens can infect fresh vegetables in different ways. Pathogenic bacteria associated with fresh vegetables can cause widespread epidemics associated with foodborne illness. The aim of this study was to assess the microbiological quality of carrot slices after treatment with aqueous extracts of Lobularia maritima (AELm) at different concentrations AELm1 (10 mg/mL), AELm2 (5 mg/mL), AELm3 (2.5 mg/mL) and AELm4 (1.25 mg/mL), and Salmonella enterica subsp. enterica serovar Enteritidis, along with vacuum packaging and storage of carrots for 7 days at 4 °C. On days 1. and 7., total viable counts (TVC), and coliforms bacteria (CB), and Salmonella count were all analysed. Microorganisms that were obtained from carrots were identified using MALDI-TOF MS Biotyper Mass Spectrometry. The total viable, coliform bacteria and Salmonella counts were varied by the group of treatment. Higher counts were found in the control group on both days. The most isolated species of bacteria were Salmonella enterica and Pantoea agglomerans on the 1. day and Klebsiella oxytoca on the 7. day. The current study adds useful information for a better understanding of how Salmonella enterica reacts to the effect of AELm and its potential use as a sustainable washing method to eliminate bacteria from freshly cut carrots.

Colonisation of hospital surfaces from low- and middle-income countries by extended spectrum β-lactamase- and carbapenemase-producing bacteria

Hospital surfaces can harbour bacterial pathogens, which may disseminate and cause nosocomial infections, contributing towards mortality in low- and middle-income countries (LMICs). During the BARNARDS study, hospital surfaces from neonatal wards were sampled to assess the degree of environmental surface and patient care equipment colonisation by Gram-negative bacteria (GNB) carrying antibiotic resistance genes (ARGs). Here, we perform PCR screening for extended-spectrum β-lactamases (blaCTX-M-15) and carbapenemases (blaNDM, blaOXA-48-like and blaKPC), MALDI-TOF MS identification of GNB carrying ARGs, and further analysis by whole genome sequencing of bacterial isolates. We determine presence of consistently dominant clones and their relatedness to strains causing neonatal sepsis. Higher prevalence of carbapenemases is observed in Pakistan, Bangladesh, and Ethiopia, compared to other countries, and are mostly found in surfaces near the sink drain. Klebsiella pneumoniae, Enterobacter hormaechei, Acinetobacter baumannii, Serratia marcescens and Leclercia adecarboxylata are dominant; ST15 K. pneumoniae is identified from the same ward on multiple occasions suggesting clonal persistence within the same environment, and is found to be identical to isolates causing neonatal sepsis in Pakistan over similar time periods. Our data suggests persistence of dominant clones across multiple time points, highlighting the need for assessment of Infection Prevention and Control guidelines.

Yeast Diversity in Honey and Pollen Samples from Stingless Bees in the State of Bahia, Brazil: Use of the MALDI-TOF MS/Genbank Proteomic Technique

(1) Background: The identification of microorganisms includes traditional biochemical methods, molecular biology methods evaluating the conserved regions of rRNA, and the molecular biology of proteins (proteomics), such as MALDI-TOF MS mass spectrometry. This work aimed to identify the biodiversity of yeasts associated with stingless bee species’ honey and pollen, Melipona scutellaris, Nannotrigona testaceicornes, and Tetragonisca angustula, from the region of São Gonçalo dos Campos-Bahia (BA) state, Brazil. (2) Methods: Cellular proteins were extracted from 2837 microbial isolates (pollen and honey) and identified via MALDI-TOF MS. The identified yeast species were also compared to the mass spectra of taxonomically well-characterized reference strains, available from the National Center of Biotechnology Information (NCBI) database. (3) Results: Nine yeast species were identified: Candida maltosa, Candida norvegica, Kazachstania telluris, Schizosaccharomyces pombe, Scheffersomyces insectosus, Meyerozyma guilliermondii, Brettanomyces bruxellensis, Kazachstania exigua, and Starmerella lactis-condensi. Nannotrigona testaceicornes pollen had the highest number of yeast colonies. The yeasts Brettanomyces bruxellensis and Kazachstania telluris showed high populations in the samples of Nannotrigona testaceicornes and Melipona scutellaris, respectively. This work shows that there is some sharing of the same species of yeast between honey and pollen from the same beehive. (4) Conclusions: A total of 71.84% of the identified species present a high level of confidence at the species level. Eight yeast species (Candida maltosa, Candida norvegica, Kazachstania telluris, Schizosaccharomyces pombe, Scheffersomyces insectosus, Meyerozyma guilliermondii, Kazachstania exigua, and Starmerella lactis-condensi) were found for the first time in the samples that the authors inspected. This contributes to the construction of new knowledge about the diversity of yeasts associated with stingless bee products, as well as to the possibility of the biotechnological application of some yeast species.

Investigation of the Microbiome of Industrial PDO Sfela Cheese and Its Artisanal Variants Using 16S rDNA Amplicon Sequencing and Shotgun Metagenomics.

Sfela is a white brined Greek cheese of protected designation of origin (PDO) produced in the Peloponnese region from ovine, caprine milk, or a mixture of the two. Despite the PDO status of Sfela, very few studies have addressed its properties, including its microbiology. For this reason, we decided to investigate the microbiome of two PDO industrial Sfela cheese samples along with two non-PDO variants, namely Sfela touloumotiri and Xerosfeli. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), 16S rDNA amplicon sequencing and shotgun metagenomics analysis were used to identify the microbiome of these traditional cheeses. Cultured-based analysis showed that the most frequent species that could be isolated from Sfela cheese were Enterococcus faecium, Lactiplantibacillus plantarum, Levilactobacillus brevis, Pediococcus pentosaceus and Streptococcus thermophilus. Shotgun analysis suggested that in industrial Sfela 1, Str. thermophilus dominated, while industrial Sfela 2 contained high levels of Lactococcus lactis. The two artisanal samples, Sfela touloumotiri and Xerosfeli, were dominated by Tetragenococcus halophilus and Str. thermophilus, respectively. Debaryomyces hansenii was the only yeast species with abundance > 1% present exclusively in the Sfela touloumotiri sample. Identifying additional yeast species in the shotgun data was challenging, possibly due to their low abundance. Sfela cheese appears to contain a rather complex microbial ecosystem and thus needs to be further studied and understood. This might be crucial for improving and standardizing both its production and safety measures.

Xanthomonas rydalmerensis sp. nov., a non-pathogenic member of Group 1 Xanthomonas.

Five bacterial isolates were isolated from Fragaria × ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus Xanthomonas. To determine their species, further analysis was conducted using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates represented members of the genus Xanthomonas but did not allow them to be classified with respect to species. Genome relatedness indices and the results of extensive phylogenetic analysis confirmed that the isolates were members of the genus Xanthomonas and represented a novel species. On the basis the minimal presence of virulence-associated factors typically found in genomes of members of the genus Xanthomonas, we suggest that these isolates are non-pathogenic. This conclusion was supported by the results of a pathogenicity assay. On the basis of these findings, we propose the name Xanthomonas rydalmerensis, with DAR 34855T = ICMP 24941 as the type strain.

Structure dependent fermentation kinetics of dietary carrot rhamnogalacturonan-I in an in vitro gut model

Plant derived dietary polysaccharides are important for gut health and have the potential to modulate the gut microbial community. Dietary rhamnogalacturonan-I obtained by enzymatic treatment of carrot pomace has shown prebiotic properties. In the present study, fermentability of carrot rhamnogalacturonan-I (cRG-I) by faecal microbiota of four donors was studied in an adapted M-SHIME® intestinal model. Despite its complex structure, cRG-I was degraded rapidly in the proximal colon compartment and fermentation became quicker and more complete during three weeks of repeated supplementation. Tracking the change in the molecular weight distribution pattern of cRG-I during the supplementation showed two main donor-dependent gut microbial fermentation strategies designated as either the general or preferential pathway. In the general fermentation pathway, different cRG-I structures were hydrolysed concomitantly, while in the preferential pathway discrete structures were sequentially fermented in a selective manner. Especially arabinan sidechains were utilized before the RG-I backbone, which correlated with an increase in Bifidobacterium longum absolute abundance over the three weeks period. MALDI-TOF MS confirmed that arabinan-, galactan- and arabinogalactan-sidechains were first to be released and degraded. Donor specific production of all SCFA increased over time with a general trend of higher levels of acetate and propionate than butyrate. Strikingly, although the host's baseline gut microbiota composition led to distinct cRG-I hydrolysis routes, the final RG-I consumption was almost complete for both routes, leading to similar metabolic profiles at the end of the three weeks treatment period.

Antifungal susceptibility profile of Candida species isolated from women with vulvovaginal candidiasis

Objectives: The objective of this study was to study the occurrence of vulvovaginal candidiasis (VVC) among women with vaginitis and to determine the antifungal susceptibility testing (AFST) pattern of Candida spp. isolated. Materials and Methods: A prospective study was conducted in a tertiary care setting. The two high vaginal swabs were collected and subjected to Gram’s stain, wet mount examination, and cultured on blood agar and sabouraud dextrose agar with chloramphenicol. The cultures were incubated at 37°C for 18–24 h. The Candida spp. was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), and AFST was performed according to the Clinical and Laboratory Standards Institute guidelines M27-A3. Statistical Analysis: The data was collected in excel sheet. The qualitative variables are presented as frequencies and percentages. The quantitative variables are presented as mean with SD and median. p value of less than 0.05 was considered statistically significant. Analysis will be done by using Epi InfoTM statistical software. Results: A total of 961 women with symptoms of vaginitis were screened. Candida spp. was isolated in 108 (11.2%) patients. The median age of affected women was 29 years, and the majority belonged to 20–30 years (60.2%). Age &lt;40 years was the significant risk factor (P = 0.016). Vaginal discharge was the most common complaint (82.4%),followed by itching (55.6%), vulvar edema, dyspareunia, and dysuria. Candida albicans was the predominant species (46.3%),followed by Candida glabrata (36.1%), Candida krusei (6.5%), and Candida tropicalis (6.5%). Ninety-six percentages of C. albicans were sensitive to fluconazole, while only 22.4% of non-albicans Candida spp (NAC) were susceptible. C. albicans showed 8% resistance to itraconazole and 8% intermediate resistance to caspofungin, whereas C. glabrata showed resistance to fluconazole, itraconazole, and caspofungin. Conclusions: The study showed a higher incidence of NAC and higher antifungal resistance leading to treatment failure. It is, hence, crucial to send fungal cultures, speciate, and perform AFST for all symptomatic patients of vulvo-vaginal candidiasis (VVC).

MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N6-methyladenosine RNA

N6-methyladenosine (m6A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m6A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m6A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m6A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m6A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64–106.93%. The present assay provides a novel platform for the analysis of site-specific m6A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.

Multidrug-Resistant Staphylococcus sp. and Enterococcus sp. in Municipal and Hospital Wastewater: A Longitudinal Study

The objective of the study was to detect multidrug-resistant Staphylococcus sp. and Enterococcus sp. isolates in municipal and hospital wastewater and to determine their elimination or persistence after wastewater treatment. Between August 2021 and September 2022, raw and treated wastewater samples were collected at two hospital and two community wastewater treatment plants (WWTPs). In each season of the year, two treated and two raw wastewater samples were collected in duplicate at each of the WWTPs studied. Screening and presumptive identification of staphylococci and enterococci was performed using chromoagars, and identification was performed with the Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS®). Antimicrobial susceptibility was performed using VITEK 2® automated system. There were 56 wastewater samples obtained during the study period. A total of 182 Staphylococcus sp. and 248 Enterococcus sp. were identified. The highest frequency of Staphylococcus sp. isolation was in spring and summer (n = 129, 70.8%), and for Enterococcus sp. it was in autumn and winter (n = 143, 57.7%). Sixteen isolates of Staphylococcus sp. and sixty-three of Enterococcus sp. persisted during WWTP treatments. Thirteen species of staphylococci and seven species of enterococci were identified. Thirty-one isolates of Staphylococcus sp. and ninety-four of Enterococcus sp. were multidrug-resistant. Resistance to vancomycin (1.1%), linezolid (2.7%), and daptomycin (8.2%/10.9%%), and a lower susceptibility to tigecycline (2.7%), was observed. This study evidences the presence of Staphylococcus sp. and Enterococcus sp. resistant to antibiotics of last choice of clinical treatment, in community and hospital wastewater and their ability to survive WWTP treatment systems.