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L. monocytogenes is considered one of the most dangerous foodborne pathogens. This study aimed to determine the occurrence of L. monocytogenes in RTE meat products from southern Poland, including serogroups and antimicrobial susceptibility, and to assess the usefulness of MALDI-TOF MS as a tool for identifying L. monocytogenes. A total of 848 production batches of RTE meat products were analyzed for L. monocytogenes. All L. monocytogenes isolates were serotyped using the multiplex PCR method, tested for antimicrobial susceptibility using the disk diffusion method and identified using the MALDI-TOF MS method. L. monocytogenes was detected in 52/848 batches of RTE meat products (6.13%). The isolates belonged to four serogroups: 17/52 (33%) isolates to IVb; 15/52 (29%) isolates to IIa; 10/52 (19%) isolates to IIc and 10/52 (19%) isolates to IIb. All isolates (52/52) showed susceptibility to the tested antimicrobials. Using MALDI-TOF MS, 10/52 isolates (19.2%) were identified at the level of secure genus identification, probable species identification; 37/52 isolates (71.2%) were identified at the level of probable genus identification; 3/52 isolates (5.8%) were incorrectly identified as L. innocua; and 2/52 isolates (3.8%) were not identified. The occurrence of L. monocytogenes in RTE meat products was low. Almost half of the analyzed isolates were L. monocytogenes of serogroups, which are most often associated with listeriosis in humans in Poland. All isolates showed susceptibility to five commonly used antimicrobials for treating listeriosis. The use of MALDI-TOF MS as a tool for the identification of L. monocytogenes indicated its limitations related to the insufficient representation of the pathogen in the reference database. [ABSTRACT FROM AUTHOR]

The phytopathogenic fungus Rhizoctonia solani distributed worldwide is one of the most serious fungal pathogens causing a wide range of plant diseases. Therefore, the present study was conducted to select endophytes from Stevia rebaudiana with biocontrol potential against Rhizoctonia solani causing sheath blight disease in maize. Out of 22 distinct bacterial endophytes from Stevia rebaudiana leaves, three bacteria showed growth inhibition of R. solani varying from 61-86% singly or in combinations. Scanning electron microscopy showed stripping of fungal hyphae leading to accumulation of debris in the presence of the antagonistic bacterial endophytes. The isolates were identified as Ochrobactrum ciceri SR1EB1,Achromobacter spanius SR1EB11 and Bacillus licheniformis SR2EB5 based on 16S rRNA gene sequencing. The strains were also tested positive for phosphate and zinc solubilization; production of auxins and ammonia; and hydrolysis of cellulose and starch. These bacterial strains exhibited disease control ranging from 66-83%, and also increased the vigour index, root length, shoot length and dry weight up to 273%, 64%, 40% and 47%, respectively, over the uninoculated control in maize grown under greenhouse conditions. The treatment OcSR1EB1+ AsSR1EB11+ BlSR2EB5 showed 2.75, 0.56, and 2.83% N, P and K content, respectively, which was the highest among all the treatments. Ochrobactrum ciceri as an endophyte from Stevia rebaudiana is reported here for the first time. The results indicated that Ochrobactrum ciceri SR1EB1,Achromobacter spanius SR1EB11 and Bacillus licheniformis SR2EB5 hold the potential to be used as biocontrol agents in controlling the sheath blight disease in maize. [ABSTRACT FROM AUTHOR]

L. monocytogenes is considered one of the most dangerous foodborne pathogens. This study aimed to determine the occurrence of L. monocytogenes in RTE meat products from southern Poland, including serogroups and antimicrobial susceptibility, and to assess the usefulness of MALDI-TOF MS as a tool for identifying L. monocytogenes. A total of 848 production batches of RTE meat products were analyzed for L. monocytogenes. All L. monocytogenes isolates were serotyped using the multiplex PCR method, tested for antimicrobial susceptibility using the disk diffusion method and identified using the MALDI-TOF MS method. L. monocytogenes was detected in 52/848 batches of RTE meat products (6.13%). The isolates belonged to four serogroups: 17/52 (33%) isolates to IVb; 15/52 (29%) isolates to IIa; 10/52 (19%) isolates to IIc and 10/52 (19%) isolates to IIb. All isolates (52/52) showed susceptibility to the tested antimicrobials. Using MALDI-TOF MS, 10/52 isolates (19.2%) were identified at the level of secure genus identification, probable species identification; 37/52 isolates (71.2%) were identified at the level of probable genus identification; 3/52 isolates (5.8%) were incorrectly identified as L. innocua; and 2/52 isolates (3.8%) were not identified. The occurrence of L. monocytogenes in RTE meat products was low. Almost half of the analyzed isolates were L. monocytogenes of serogroups, which are most often associated with listeriosis in humans in Poland. All isolates showed susceptibility to five commonly used antimicrobials for treating listeriosis. The use of MALDI-TOF MS as a tool for the identification of L. monocytogenes indicated its limitations related to the insufficient representation of the pathogen in the reference database.

O diagnóstico das enfermidades transmitidas por micobactérias em animais domésticos e selvagens representa um grande desafio à medicina veterinária. Embora o Mycobacterium bovis, seja a espécie de maior interesse por causar a tuberculose, as micobactérias não tuberculosas (MNT), também podem causar graves infecções, denominadas micobacterioses, em animais e no homem. As doenças micobacterianas caracterizam-se pelo desenvolvimento granulomatoso crônico e progressivo. O tempo de geração das espécies de crescimento lento pode chegar até vinte horas, fazendo com que seu isolamento em meios de cultura tradicionais, possa demorar até 90 dias para espécies de crescimento lento e aproximadamente seis dias para espécies de crescimento rápido. Além de fastidiosas, as micobactérias possuem parede celular rica em lipídeos e ácidos micólicos, o que dificulta a extração de DNA, interferindo em muitos métodos moleculares utilizados na tentativa de acelerar o diagnóstico. Embora a diferenciação entre as espécies tenha avançado nos últimos anos com a utilização de métodos moleculares mais específicos, em espécies geneticamente muito próximas, este processo ainda é muito desafiador, estimulando-se o desenvolvimento de novas tecnologias. As metodologias de espectrometria de massas como o MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry), têm sido utilizadas como ferramenta de identificação microbiológica em laboratórios clínicos, indústrias alimentícias e hospitais. Neste artigo, serão discutidos os principais aspectos relacionados ao uso desta ferramenta para o diagnóstico de micobactérias provenientes de amostras clínicas de animais.

In this study, it was aimed to isolate, identify, and determine some starter culture characteristics of lactic acid bacteria from yoghurts produced by traditional methods, collected from five provinces in the Western Black Sea Region. Identification of strains was performed with the MALDI-TOF MS Biotyper system. Of the 84 isolates isolated, 2 were identified as Lactobacillus helveticus, 2 as Lactiplantibacillus plantarum, 3 as Limosilactobacillus fermentum, 1 as Enterococcus faecalis and 76 as Lactobacillus delbrueckii. The isolates were found to be susceptible to erythromycin, teicoplanin, streptomycin, rifampicin, ampicillin, clindamycin, cefotaxime, chloramphenicol, tetracycline, and vancomycin, and resistant to nalidixic acid, ciprofloxacin, ofloxacin, gentamicin and trimethoprim sulfamethoxazole. It was determined that the isolates showed antimicrobial activity against Salmonella Typhimurium and E. coli O157:H7, but the antimicrobial effect was low against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. The acidity produced by 8 of the isolates was found to be between 0.9 and 0.95 %. In addition, it was determined that 74 isolates produced diacetyl. Proteinase activity and resistance to phages of 26 strains with high starter potential were investigated. It was determined that 5 of the strains did not show proteinase activity and according to the phage resistance test results, 8 of the isolates were sensitive to Ф709-X1 phage, and 12 Lactobacillus delbrueckii isolates were resistant to 15 different bacteriophages used in the study. According to the results of the study, it was determined that the isolated cultures have the potential to be used as a starter culture in terms of the investigated characteristics. [ABSTRACT FROM AUTHOR]

The present study introduces a non-destructive method of analysis by MALDI TOF Mass spectrometry to identify the natural dyes in four Iranian archaeological carpets, date to the 17-18th century, attributed to Sarband and Tabriz cities. The study aims to unveil a method of analysis that may not have been used in dye analysis and to identify the samples taken from these carpets, which have reached a high rate of deterioration, and need a rapid intervention to be preserved. The study proved using natural indigo in blue, light blue, navy, and pale navy in the four carpets. The use of natural indigo (as a blue dye) has also been proved in oily, light oily, and greenish-blue samples in the four carpets along with weld and Persian berries dyes in the light oily color sample in Tabriz II. The madder and cochineal pigments were proven to be used in dyeing with the coppery red color in the first Sarband carpet. The madder and yellow dye were used for dyeing with coppery red color in the second Tabriz carpet. The madder dye has been proven to be used in dyeing with the coppery red and brick red in the third/fourth carpet. The results showed the use of weld dye in the beige color in the third/fourth carpet, while, no dye was detected in the beige color in the first and second carpet, as well as the pale yellow color in the second carpet. The examined Mass method proved a non-invasive, very fast, and high ability in identifying many dyes' compounds, without any dyes extraction or sample preparation. The method was unable to distinguish between compounds of the same molar mass, e.g. indigotin and indirubin compounds in indigo. So far, and according to the followed methodologies, the method was detected relatively few numbers of dyes' compounds, so, the type of dye isn't confirmed due to the presence of the same compound in more than one dye. Due to its many features; the study recommends more research about this method to unveil the reasons for the less detected compounds, and whether it can be overcome.

The purpose of this study was to conduct a comparative analysis of the composition of the dominant groups of vaginal microorganisms in healthy pregnant women and pregnant women infected with HPV using a microbiological culture-based method. The MALDI TOF MS method and 16S rRNA gene fragment sequencing were used to identify microorganisms isolated from healthy pregnant women (n=32) and pregnant women infected with HPV (n=24). It was found that vaginal secretion samples from both groups contained bacteria of 4 phyla: Bacillota, Actinomycetota, Pseudomonadota, Bacteroidota , and Ascomycota fungi. The most common microbial community in healthy pregnant women being CST I (p=0.0007), and CST V in pregnant women infected with HPV (p=0.0001). At the genus level, a total of 25 taxa were found in all samples, with Lactobacillus being the dominant genus overall. Escherichia (p<0.0001) and Prevotella (p=0.0001) concentrations were higher in HPV infected patients. When calculating the Pearson correlation coefficient for the phyla, it was found that Bacillota correlated negatively with HPV genotypes 16 and 51 (p≤0.05), but positively with HPV genotype 59 (p≤0.05), just like Actinomycetota (p≤0.05). Bacteroidota correlated positively with HPV genotype 56 (0.001

Veillonella parvula is a non-motile gram-negative coccus that forms part of the normal microbiota in several body sites and which has been rarely isolated as cause of infections in human population, particularly in bacteremias. Here we give the overview of characteristics of genus Veillonella and the summary of its role in infections, particularly in bacteremia. We additionally report two patients with bacteremia due to V. parvula. Two sets of blood cultures of each patient yielded a pure culture of an anaerobic microorganism identified as V. parvula by MALDI-TOF MS, and confirmed by 16S rRNA gene sequencing. The two patients were male and one of them had risk factors for anaerobic bacteremia. The isolates were susceptible to most antibiotics and the outcome was successful in both patients. Bacteremia due to V. parvula is still rare. MALDI-TOF MS appear to be an excellent tool for the correct identification of these species. • Veillonella spp are uncommon causes of bacteremia. • Veillonellaceae plays a key role in oral biofilm development. • MALDI-TOF MS is a good diagnostic tool for these bacteria. • Antimicrobial susceptibility testing should be performed in cases of bacteremia. [ABSTRACT FROM AUTHOR]

Cardiobacterium hominis is a member of the HACEK group of bacteria, responsible for infective endocarditis, mainly in patients with damaged or prosthetic valves. The low virulence of this organism can explain the insidious presentation and subacute or chronic progression of C. hominis infective endocarditis. Here, a 41-year-old man with a past history of surgery for a Waldhausen type aortic coarctation was hospitalised with dyspnea and chest pains revealing an acute pulmonary oedema, without fever. Transesophageal echocardiography indicated a 20 mm vegetation on biscuspid aortic valve. Six sets of blood culture were positive with Cardiobacterium hominis. In case of lack of fever, the diagnosis of infectious endocarditis is difficult because other symptoms are non-specific and biological markers of inflammatory syndrome are quiet or non-existent. This is the first case of C. hominis infectious endocarditis with a clinical presentation of acute pulmonary oedema in the literature. We report here an apyretic pulmonary oedema revealing C. hominis endocarditis and a review of the literature on apyretic infective endocarditis due to C. hominis.

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The review reflects the current state of the working in collection and presents the directions of its improvement within the framework of the carried out certification of the collection of pathogenic Burkholderia spp. of Volgograd Research Institute for Plague Control. The ways of modernization of existing methods of conservation, optimization of modern methods of phenotypic and molecular-genetic characteristics of strains of pathogenic microorganisms are considered, and an information cataloging system with the formation of a universal database is proposed.

Identification of moulds is crucial for the clinical management of patients. The goal of this study was to evaluate the new ID-FUNGI plate (IDFP) for the identification of moulds by MALDI Biotyper. IDFP was compared with Sabouraud with gentamicin and chloramphenicol plate (SAB) for the identification of 80 moulds from respiratory samples and eight reference strains. With the direct transfer method, species identification rose from 6% with SAB to 68% with IDFP using score cut-off 2 and from 20 to 75% using cut-off 1.7 (p < 0.001). Our study highlights that the new IDFP improves mycological diagnostic and workflow in laboratories. [ABSTRACT FROM AUTHOR]

Bovine mastitis is a prevalent disease that causes serious economic problems globally in the dairy industry. Staphylococcus aureus is an important pathogen of bovine mastitis. This study was conducted to characterize S. aureus isolates from clinical bovine mastitis cases in large-scale dairy herds in China. S. aureus was isolated from 624 clinical mastitis cases and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 62 S. aureus isolates were obtained. Cluster analysis, genetic diversity, quantification of biofilm formation, antimicrobial resistance, and detection of virulence genes were performed on these isolates of S. aureus. Eight isolates harbored the mecA gene and were sensitive to oxacillin. MALDI-TOF MS cluster analysis revealed that the 62 isolates were divided into three major clusters (I, II, III) and eight main groups (A–H) at the distance level of 700. The agr II was the most prevalent (56.5%). The 62 S. aureus isolates were assigned to seven spa types. The most common spa type was t529(58.1%), followed by t2196 (14.5%), t518 (14.5%), t571(6.5%), t034 (3.2%), t2734 (1.6%), and t730 (1.6%). Five STs were identified from seven representative isolates as follows: ST630/CC8, ST97/CC97, ST50, ST398, and ST705. All isolates had the ability to form biofilm. Antimicrobial resistance was most frequently observed to ciprofloxacin (29%), followed by penicillin (24.2%), and streptomycin (9.6%). All isolates harbored the fnbA, clfB (100%), icaA, and icaD genes. This study provides the basis for the development of bovine mastitis prevention program on large-scale dairy farms.

The case of a 32-year-old woman is reported, who was affected by a persisting wound infection caused by after an accident in the Mediterranean Sea. Besides the clinical case, microbiological characteristics based on the phenotypic and genotypic description of the isolate (including whole genome data) are presented and discussed.

The emergence and spread of the novel mobile Tet(X) tetracycline destructases confer high-level tigecycline and eravacycline resistance in Escherichia coli and Acinetobacter spp. and pose serious threats to human and animal health. Therefore, a rapid and robust Tet(X) detection assay was urgently needed to monitor the dissemination of tigecycline resistance. We developed a rapid and simple assay to detect Tet(X) producers in Gram-negative bacteria based on matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). This MALDITet(X) test was based on the inactivation of tigecycline by a Tet(X)-producing strain after a 3-h incubation of bacterial cultures with tigecycline. Culture supernatants were analyzed using MALDI-TOF MS to identify peaks corresponding to tigecycline (586 ± 0.2 m/z) and a tigecycline metabolite (602 ± 0.2 m/z). The results were calculated using the MS ratio [metabolite/(metabolite + tigecycline)]. The sensitivity of the MALDITet(X) test with all 216 test strains was 99.19%, and specificity was 100%. The test can be completed within 3 h. Overall, the MALDITet(X) test is an accurate, rapid, cost-effective method for the detection of Tet(X)-producing E. coli and Acinetobacter spp. by determining the unique peak of an oxygen-modified derivative of tigecycline.

Abstract Background Yersinia enterocolitica is widespread within the humans, pigs and wild boars. The low isolation rate of Y. enterocolitica from food or environmental and clinical samples may be caused by limited sensitivity of culture methods. The main goal of present study was identification of presumptive Y. enterocolitica isolates using MALDI TOF MS. The identification of isolates may be difficult due to variability of bacterial strains in terms of biochemical characteristics. This work emphasizes the necessity of use of multiple methods for zoonotic Y. enterocolitica identification. Results Identification of Y. enterocolitica isolates was based on MALDI TOF MS, and verified by VITEK® 2 Compact and PCR. There were no discrepancies in identification of all human’ and pig’ isolates using MALDI TOF MS and VITEK® 2 Compact. However three isolates from wild boars were not decisively confirmed as Y. enterocolitica. MALDI TOF MS has identified the wild boar’ isolates designated as 3dz, 4dz, 8dz as Y. enterocolitica with a high score of matching with the reference spectra of MALDI Biotyper. In turn, VITEK® 2 Compact identified 3dz and 8dz as Y. kristensenii, and isolate 4dz as Y. enterocolitica. The PCR for Y. enterocolitica 16S rDNA for these three isolates was negative, but the 16S rDNA sequence analysis identified these isolates as Y. kristensenii (3dz, 4dz) and Y. pekkanenii (8dz). The wild boar’ isolates 3dz, 4dz and 8dz could not be classified using biotyping. The main bioserotype present within pigs and human faeces was 4/O:3. It has been shown that Y. enterocolitica 1B/O:8 can be isolated from human faeces using ITC/CIN culturing. Conclusion The results of our study indicate wild boars as a reservoir of new and atypical strains of Yersinia, for which protein and biochemical profiles are not included in the MALDI Biotyper or VITEK® 2 Compact databases. Pigs in the south-west Poland are the reservoir for pathogenic Y. enterocolitica strains. Four biochemical features included in VITEK® 2 Compact known to be common with Wauters scheme were shown to produce incompatible results, thus VITEK® 2 Compact cannot be applied in biotyping of Y. enterocolitica.

Aim. Identification of obtained in host-simulated conditions immunogenic proteins of isogenic variants of Bacillus anthracis 575/122. Materials and methods. We used culture filtrate of isogenic variants of B. anthracis 575/122: R02 (pXOL pXO2+); R01 (pXO1+ pXO2‘); R00 (pXOL pX02~), obtained in host-simulated conditions. In the one-dimensional polyacrylamide gel electrophoresis and immunoblotting with hyperimmune serums immunodominant proteins, that have been identified in MALDI TOF MS. Results. Immunoblotting revealed proteins with molecular masses in range 97 - 14.1 kDa. 90 kDa protein from strain B. anthracis 575/122 R01 in MALDI TOF MS was identified as protective antigen with 85.810 kDa. Protein with molecular mass 60 kDa was identified as GMP synthase with molecular mass 57.239 kDa. In the culture filtrates of three strains two common antigen were identified: protein with molecular mass 97 kDa, identified as B. anthracis EA 1 with molecular mass 91.361 kDa protein and 45 kDa protein as enolase B. anthracis with molecular mass 46.418 kDa. Conclusion. Thus, the conditions that simulate the host can promote the production of immunodominant proteins of B. anthracis. The data about molecular-weight characteristics of protective antigen and EA 1 protein as well as some of proteases of B. anthracis are confirmed by the MALDI TOF MS. The results can be used for isolation of these proteins to improve the diagnostic and vaccine preparations.

Listeria monocytogenes, yaşlılarda ve predispozan koşulları olan bağışıklık sistemi baskılanmış hastalarda enfeksiyona neden olabilecek önemli bir patojendir. Bu bakteri, bağışıklık sistemi baskılanmış kişilerde hayatı tehdit eden bakteriyemi ve meningoensefalitin önemli bir nedeni olabilir. Bu raporda, larenks adenoid kistik karsinomu olan 39 yaşındaki bir kadın hastada L. monocytogenes'in etken olduğu sepsis ve menenjit olgusu tanımlandı. L. monocytogenes'in tür düzeyinde tanımlanması matrix-assisted laser desorption and ionization timeof- flight mass spectrometry (MALDI-TOF MS, Bruker, Almanya) ile yapıldı. Hasta tedavide seftriaksondan ampisiline erken geçiş ile tedavi edildi. Bu rapor, Listeria sepsis ve meninjitinin erken saptanmasının ve tedavisinin, daha iyi bir prognoz elde edilmesindeki önemini işaret etmektedir. [ABSTRACT FROM AUTHOR]

The emergence and spread of the novel mobile Tet(X) tetracycline destructases confer high-level tigecycline and eravacycline resistance in Escherichia coli and Acinetobacter spp. and pose serious threats to human and animal health. Therefore, a rapid and robust Tet(X) detection assay was urgently needed to monitor the dissemination of tigecycline resistance. We developed a rapid and simple assay to detect Tet(X) producers in Gram-negative bacteria based on matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). This MALDITet(X) test was based on the inactivation of tigecycline by a Tet(X)-producing strain after a 3-h incubation of bacterial cultures with tigecycline. Culture supernatants were analyzed using MALDI-TOF MS to identify peaks corresponding to tigecycline (586 ± 0.2 m/z) and a tigecycline metabolite (602 ± 0.2 m/z). The results were calculated using the MS ratio [metabolite/(metabolite + tigecycline)]. The sensitivity of the MALDITet(X) test with all 216 test strains was 99.19%, and specificity was 100%. The test can be completed within 3 h. Overall, the MALDITet(X) test is an accurate, rapid, cost-effective method for the detection of Tet(X)-producing E. coli and Acinetobacter spp. by determining the unique peak of an oxygen-modified derivative of tigecycline. [ABSTRACT FROM AUTHOR]

Urinary tract infections are one of the most common bacterial infections and rapid diagnosis of the infection is essential for appropriate antibiotic therapy. The goal of our study was to identify urinary pathogens directly by MALDI-TOF MS and to perform antibiotic susceptibility tests in order to shorten the period spent for culturing. Urine samples submitted for culture to the Clinical Microbiology Laboratory were enrolled in this study. Urine samples were screened for leukocyte and bacteria amount by flow cytometry. Samples with bacterial load of 106–107/mL were tested directly by MALDI-TOF MS and antibiotic susceptibility tests (AST) were performed. In total, 538 positive urine samples were evaluated in our study. MALDI-TOF MS identified the microorganism directly from the urine sample in 91.8% of these samples and the concordance rate of conventional identification and direct detection was 95.8% for Gram-negatives at the genus and species level. Escherichia coli (n :401) was the most frequently isolated microorganism, followed by Klebsiella pneumoniae (n :57). AST results were generated for 111 of these urine samples and the concordance was 90% and 87% for E. coli and K. pneumoniae , respectively. Our results showed that screening of urine samples with flow cytometry to detect positive samples and identification of uropathogens directly by MALDI-TOF MS with an accuracy of over 90% can be a suitable method particularly for Gram-negative bacteria in clinical microbiology laboratories. [ABSTRACT FROM AUTHOR]

Bacground: Microbial Identification was done by phenotypic methods. VITEK-2 and Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are now being increasingly used in laboratories. Objectives: To compare and evaluate the usefulness of MALDI-TOF MS and VITEK-2 in routine microbial identification. Methods: The performances of MALDI-TOF MS and VITEK 2 were compared for identifying microorganisms. Results: MALDI-TOF MS and VITEK-2 correctly identified 96 % (96/100) and 97% (97/100) of the isolates upto the genus level. Conclusion: MALDI TOF MS and VITEK -2 gave comparable identification and error rates. The rapid reduction in turnaround time with MALDI TOF is a significant game-changer in the field of clinical microbiology. [ABSTRACT FROM AUTHOR]

MALDI TOF MS-based microbial identification significantly lowers the operational costs because of minimal requirements of substrates and reagents for extraction. Therefore, it has been widely used in varied applications such as clinical, food, military, and ecological research. However, the MALDI TOF MS method is laced with many challenges including its limitation of the reference spectrum. This review briefly introduces the background of MALDI TOF MS technology, including sample preparation and workflow. We have primarily discussed the application of MALDI TOF MS in the identification of microorganisms. Furthermore, we have discussed the current trends for bioaerosol detection using MALDI TOF MS and the limitations and challenges involved, and finally the approaches to overcome these challenges.

The quantitative determination of small molecules by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) was always challenging, due to low sensitivity and interferences of matrix and other contaminants on sample plates. Here we report a disposable paper-array (PA) plate in MALDI TOF MS analysis for quantitative analysis of serum free fatty acids (FFAs) with less interference and higher sensitivity, compared with conventional stainless steel (SS) plate. The disposable PA plate was low cost and environmentally friendly. In particular, it was more sensitive in FFAs detection with ~8–34-fold sensitivity increase for eight FFAs as compared to SS plate. Eight serum FFAs in gout patients were investigated using this disposable PA plate. The quantitative results showed good linearity of all eight FFAs between 0.1 and 2.5 mM with correlation coefficients between 0.994 and 0.999 and limits of detection (LODs) between 8.6 and 104.2 μM. Mann-Whitney U test, principal component analysis (PCA) and random forest categorizer allowed the clear separation of gout patients from healthy controls, according to eight FFAs levels in serum detected by disposable PA plate based MALDI TOF MS. Here we report a disposable paper-array (PA) plate in MALDI TOF MS analysis for the quantitative analysis of serum free fatty acids (FFAs) with less interference and higher sensitivity than conventional stainless steel (SS) plate. Mann-Whitney U test, principal component analysis (PCA) and random forest categorizer allowed the clear separation of the gout patients from the healthy controls according to eight FFAs levels in serum detected by disposable PA plate based MALDI TOF MS. Image 1 • A low-cost and environmentally friendly disposable paper plate was developed. • The detection of free fatty acids had less interference and higher sensitivity. • Eight serum FFAs in gout patients were investigated using the disposable PA plate. • Categorizers allowed the separation of the gout patients from the healthy controls. [ABSTRACT FROM AUTHOR]

The most commonly used probiotic bacteria belong to the genus Lactobacillus, being regarded as beneficial for poultry health and production. However, commercial probiotics do not always ensure both expected effects. In order to improve the utility properties of new preparations, the selection of new probiotic candidates should be made on the basis of the performance of the species within the poultry digestive tract. The aim of this study was to isolate and identify lactobacilli from poultry intestines, and to select probiotic candidates for subsequent in vivo trials. Digesta from 18 poultry specimens were collected, serially diluted, plated onto Wilkins-Chalgren anaerobe agar supplemented with 30% of rumen fluid and onto De Man, Rogosa and Sharpe agar plates, and incubated at 37 °C for 48 h under anaerobic conditions. Isolated colonies were subjected to Gram staining and catalase reaction. They were then pre-identified using matrixassisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). Forty-four Lactobacillus strains belonging to 16 species were identified and subjected to evaluations of survival under simulated gastrointestinal conditions, autoaggregation and hydrophobicity. Most of the screened Lactobacillus reuteri strains as well as individual strains of L. acidophilus, L. amylovorus, L. gallinarum, L. ingluviei, L. johnsonii, L. oris, L. salivarius, L. saerimneri, and L. vaginalis showed high survival rates under gastrointestinal tract conditions and good surface properties. The results suggest their potential for further testing as probiotic candidates in in vivo trials. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption ionization high-resolution time-of-flight mass spectrometry (MALDI HR TOF MS) is a powerful tool for the molecular characterization of industrial polymers. However, accurate mass determination and resolution of isobaric ions are possible for oligomer samples only typically below m/z 3000. To cut long polymer chains into oligomers suitable for high-resolution mass spectrometry, we propose a simple "on-plate" alkaline degradation of polyesters as a sample pretreatment technique prior to the MALDI TOF MS measurement. This pretreatment can be performed on a MALDI target using a small amount of sample (μg or less) and 1 μL of alkaline reagent by simple pipetting. Informative mass spectra in the oligomeric mass range are successfully recorded but complicated by the variation of end-groups and the copolymeric composition of the degradation products. Data processing is assisted by a series of advanced Kendrick mass defect (KMD) analyses recently proposed by the authors to plot visually understandable two-dimensional maps. On-plate degradation pretreatment, high-resolution MALDI TOF MS measurements, and advanced KMD analyses are innovatively combined for the compositional characterization of bacterial poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) and industrial poly(ethylene terephthalate) samples.ᅟ [ABSTRACT FROM AUTHOR]

Staphylococcus aureus remains a major health problem responsible for many epidemic outbreaks. Therefore, the development of efficient and rapid methods for studying molecular profiles of S. aureus strains for its further typing is in high demand. Among many techniques, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI TOF MS) represents a timely, cost-effective, and reliable strain typing approach, which is still rarely used due to insufficient knowledge about the impact of sample preparation and analysis conditions on the molecular profiles and strain classification efficiency of S. aureus. The aim of this study was to evaluate the effect of the culture conditions and matrix type on the differentiation of molecular profiles of various S. aureus strains via the MALDI TOF MS analysis and different computational methods. The analysis revealed that by changing the culture conditions, matrix type, as well as a statistical method, the differentiation of S. aureus strains can be significantly improved. Therefore, to accelerate the incorporation of the MALDI-based strain typing in routine laboratories, further studies on the standardization and searching of optimal conditions on a larger number of isolates and bacterial species are of great need.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer’s disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method’s applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research.ᅟ [ABSTRACT FROM AUTHOR]

Treating cancer with chemotherapy is accompanied with side effects and drug resistance by cancer cells which call for alternative anticancer agents such as cationic antimicrobial peptides. In specific, antifungal peptides were chosen for cytotoxicity analysis due to their deteriorating effects on fungi (eukaryotic cells). Novel peptides were isolated and analysed from red radish seeds. Water soaked seeds were ground with phosphate buffer (pH 7) and the antifungal assay was carried out and an inhibition zone of 0.5±0.2 mm was noticed for 50 μl of the extract at 1 mg/ml concentration. The study was extended further, by digestion with papain, trypsin and pepsin with appropriate buffer and pH of which papain digests of red radish seeds produced a zone of inhibition of 1.00±0.2 mm at 500 μg/ml concentration, approximately. This sample was processed with a 3 kDa cut off membrane and the filtrate was confirmed for its antifungal activity (MIC 32 μg/ml). Further, for size confirmation, the sample was subjected to MALDI-TOF-MS analysis, and a peak was obtained at 1680 Da (1.68 kDa). To check the anticancer activity of the antifungal peptides obtained, the sample was tested on HeLa cell lines by method of MTT Assay and significant cell death was obtained at MIC 12.5 μg/ml which increased with higher concentrations of the sample. Thus, it is confirmed that red radish seed extract digested with papain is effective in inhibiting the growth of both fungal and cancer cell lines making them a good therapeutic agent against cancer. [ABSTRACT FROM AUTHOR]

Invertebrate pests and parasites of humans, animals, and plants continue to cause serious diseases and remain as a high treat to agricultural productivity and storage. The rapid and accurate species identification of the pests and parasites are needed for understanding epidemiology, monitoring outbreaks, and designing control measures. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as a rapid, cost effective, and high throughput technique of microbial species identification in modern diagnostic laboratories. The development of soft ionization techniques and the release of commercial pattern matching software platforms has resulted in the exponential growth of applications in higher organisms including parasitology. The present review discusses the proof-of-principle experiments and various methods of MALDI MS profiling in rapid species identification of both laboratory and field isolates of pests, parasites and vectors.

The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential.

Five new catalysts are prepared from dibutyltin oxide and catechol (HCa), 2,3-dihydroxynaphthalene (NaCa), 4- tert-butyl catechol (BuCa), 4-cyano catechol (CyCa), and 4-benzoyl catechol (BzCa), but only BuCa gives useful results. When benzyl alcohol is used as an initiator, linear chains having benzyl ester end groups are formed in a slow polymerization process. In contrast to cyclic or noncyclic dibutyltin bisalkoxides, neat BuCa yields cyclic poly( l-lactide)s via a fast ring-expansion polymerization. Under certain conditions, a high-melting crystalline phase ( Tm = 191 °C) is obtained. At 160 °C and short reaction times even-numbered cycles are slightly prevailing, but, surprisingly, at 120 °C, odd-numbered cycles are predominantly formed. These results definitely prove that a ring-expansion mechanism is operating. [ABSTRACT FROM AUTHOR]

Mycotoxins are toxic substances produced by some microscopic fungi such as Aspergillus, Fusarium and Penicillium sp. These species commonly contaminate foods and feeds. Therefore, the quick and accurate detection is very necessary. In this study, a six mycotoxins, concretely: aflatoxin B1, citrinin, deoxynivalenol, zearalenone, T2-toxin, and griseofulvin were detected by Matrix Assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS). In addition, sodium adducts were observed in all tested mycotoxins. All mycotoxins were ionizated by HCCA matrix. Our results showed that it is possible to detect selected mycotoxins by MALDI-TOF MS Microflex LT in linear positive ion mode. [ABSTRACT FROM AUTHOR]

Summary Saprochaete capitata may cause uncommon severe infections, especially in immunocompromised patients. Here, we describe a rare case of urinary tract infection by S . capitata in a chronic kidney disease and diabetes mellitus patient, which occur during anidulafungin therapy. Mycological examinations of urine were positive to S . capitata identified by mass spectrometry and confirmed by ITS sequencing. Minimum inhibitory concentration (MIC) of the isolate for amphotericin B, fluconazole, itraconazole, voriconazole and, anidulafungin were 2, 16, 1, 1, and 8 μg/mL, respectively. Presence of S . capitata infection was not known. Clinicians should be aware about these rare opportunistic fungal pathogens, particularly those with intrinsic or variable resistance to antifungals including echinocandins. [ABSTRACT FROM AUTHOR]

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder ( Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either 'no reliable identification' or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen. [ABSTRACT FROM AUTHOR]

Murdochiella asaccharolytica is a Gram-positive anaerobic coccus firstly isolated from wound specimens, since then, it was very rarely isolated from human infections. We report the first case of bloodstream infection due to this bacterium in a 90 years-old-man. The patient had a history of B-cell lymphoma and presented with pain and induration of the base of penis along with fever. A blood culture was obtained yielding M. asaccharolytica in pure culture. Only resistance to clindamycin was demonstrated. Treatment with metronidazole was started and the clinical evolution improved with antimicrobial treatment, being discharged after 6 days. • Murdochiella was firstly associated with a wound infection. • This is the first case of bacteremia caused by M. asaccharolytica. • MALDI-TOF MS is useful in the diagnosis of new anaerobic pathogens. • It is strongly recommended to report these new infections. [ABSTRACT FROM AUTHOR]

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1 and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analysed via MALDI- TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g. malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g. phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae.

We here show adequate species identification for bacterial isolates of the genus Nocardia spp. through VITEK mass spectrometry. Application of a specific sample preparation method in combination with a robust matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) database leads to 94% accurate identification to the species level on a set of 164 isolates. The possibility to identify Nocardia spp. using MALDI-TOF MS will be available in the next release of VITEK MS update (IVD Version 3.0). [ABSTRACT FROM AUTHOR]

Mycobacterial identification using MALDI-ToF MS (MALDI) has been hindered by inadequate extraction methods. Adaptive Focused Acoustics™ uses concentrated ultrasonic energy to achieve cellular disruption. Using this technology, we developed a rapid mycobacterial inactivation/protein extraction method for MALDI-based identification. Agreement for identification to the species level versus conventional identification was stratified by log confidence cut-offs of ≥2.0, ≥ 1.8, or ≥1.7. A total of 182 mycobacterial isolates were tested. Complete inactivation of all species/strains was achieved after 2 min. Using a log confidence cut-off of ≥2.0, overall agreement for the commercial method (CM) was 41.7% versus 66.7% for the novel method (NM). For the CM, agreement increased to 66.7% and 83.3% using log confidence cut-offs of ≥1.8 and ≥1.7, respectively; for the NM, agreement was 100% for both cut-offs with all isolates. With no alteration to the existing database, overall agreement for the NM was 83.4%, largely due to low scores for clinical isolates of M. chelonae and M. mucogenicum. Addition of spectra from a single clinical strain of each species to the existing database increased overall agreement to 93.1%. [ABSTRACT FROM AUTHOR]

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory. [ABSTRACT FROM AUTHOR]

Abstract Bacteroides pyogenes can cause infections in humans. We describe a case of bloodstream infection caused by Bacteroides denticanum that probably originated from a dog bite. MALDI-TOF MS misidentified this new species as B. pyogenes. Subsequent analysis using the 16S rRNA sequencing approach identified the species as B. denticanum. Highlights • Bacteroides denticanum bloodstream infection was described. • MALDI TOF MS misidentified Bacteroides denticanum as Bacteroides pyogenes. • 16S rRNA sequencing is needed for distinguishing between these related species. [ABSTRACT FROM AUTHOR]

We report an uncommon case of bacteremia due to Lancefieldella parvula isolated in pure culture. We present a 72 year-old-man affected with cancer admitted with abdominal pain due to a parietal rupture of the urinary bladder. After surgical treatment, the patient developed fever and two sets of blood cultures yielded a pure culture of an anaerobic microorganism identified as L. parvula (formerly Atopobium parvulum) by MALDI-TOF MS, and confirmed by 16S rRNA gene sequencing. The isolate was susceptible to all antibiotics and the outcome was finally successful. Bacteremia due to L. parvula is an uncommon disease and, in that case, MALDI-TOF MS was an useful tool for the initial identification. • Lancefieldella parvula has been rarely associated with bacteremia. • The patient had some risk factors for anaerobic bacteremia. • MALDI-TOF MS provided a correct species identification. • The isolate showed susceptibility to all antimicrobials. [ABSTRACT FROM AUTHOR]