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Objectives: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it.Methods: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared.Results: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2–25]), reproducibility between technical replicates (range = [55%–86%]), and measurement error (range = [147 parts per million (ppm)–588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values Discussion: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

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

Although Weissella cibaria and W. confusa are essential food-fermenting bacteria, they are also opportunistic pathogens. Despite these species being commercially crucial, their taxonomy is still based on inaccurate identification methods. In this study, we present a novel approach for identifying two important Weissella species, W. cibaria and W. confusa, by combining matrix-assisted laser desorption/ionization and time-of-flight mass spectrometer (MALDI-TOF MS) data using machine-learning techniques. After on- and off-plate protein extraction, we observed that the BioTyper database misidentified or could not differentiate Weissella species. Although Weissella species exhibited very similar protein profiles, these species can be differentiated on the basis of the results of a statistical analysis. To classify W. cibaria, W. confusa, and non-target Weissella species, machine learning was used for 167 spectra, which led to the listing of potential species-specific mass-to-charge (m/z) loci. Machine-learning techniques including artificial neural networks, principal component analysis combined with the K-nearest neighbor, support vector machine (SVM), and random forest were used. The model that applied the Radial Basis Function kernel algorithm in SVM achieved classification accuracy of 1.0 for training and test sets. The combination of MALDI-TOF MS and machine learning can efficiently classify closely-related species, enabling accurate microbial identification.

Carbapenemase resistance in Enterobacterales is a global public health problem and rapid and effective methods for detecting these resistance mechanisms are needed urgently. Our aim was to evaluate the performance of a MALDI-TOF MS-based “Klebsiella pneumoniae carbapenemase” (KPC) detection protocol from patients’ positive blood cultures, short-term cultures, and colonies in healthcare settings. Bacterial identification and KPC detection were achieved after protein extraction with organic solvents and target spot loading with suitable organic matrices. The confirmation of KPC production was performed using susceptibility tests and blaKPC amplification using PCR and sequencing. The KPC direct detection (KPC peak at approximately 28.681 Da) from patients’ positive blood cultures, short-term cultures, and colonies, once bacterial identification was achieved, showed an overall sensibility and specificity of 100% (CI95: [95%, 100%] and CI95: [99%, 100%], respectively). The concordance between hospital routine bacterial identification protocol and identification using this new methodology from the same extract used for KPC detection was ≥92%. This study represents the pioneering effort to directly detect KPC using MALDI-TOF MS technology, conducted on patient-derived samples obtained from hospitals for validation purposes, in a multi-resistance global context that requires concrete actions to preserve the available therapeutic options and reduce the spread of antibiotic resistance markers.

Soft rot disease in potato is a major problem in fields and warehouses all over the world. Although it is known that bacteria from the genera Pectobacterium and Dickeya are the main causative agents of soft rot diseases, recent studies indicate the involvement of pectinolytic Bacillus and Paenibacillus in this disease. In the present research, samples of potato with soft rot symptoms were collected from eight governorates of Tunisia. Two hundred and seventy bacterial isolates were acquired from tubers. Twenty of the isolated strains indicted pectinolytic activity by forming deep cavities on crystal violet pectate medium. All pectinolytic isolates were able to macerate potato tuber tissue. Phenotypic characterisation showed that these isolates were Gram-positive bacilli, exhibiting pectinolytic, cellulolytic, proteolytic and amylolytic activity. The majority of the isolates indicated swimming and swarming motility. The application of API test, MALDI-TOF MS and 16S rDNA sequencing allowed for the assignment of nineteen of the tested isolates to the species Bacillus pumilus and one to the species Paenibacillus amyloliticus. To the best of our knowledge, this is the first report of soft rot in potato caused by pectinolytic B. pumilus in Tunisia.

Introduction: Resistance to carbapenems due to the co-production of NDM and ESBL or NDM and KPC is increasing. Therefore, combined therapy with aztreonam (ATM) plus ceftazidime/avibactam (CZA) has been recommended. Then, it is necessary to develop and evaluate fast and simple methods to determine synergism in vitro in microbiology laboratories. Objective: To develop a method to determine the synergism of ATM and CZA by MALDI-TOF MS (SynMALDI). Method: Klebsiella pneumoniae (n = 22) isolates with blaNDM and/or blaKPC genes were tested. The time–kill curve assay was performed for four isolates (three positives for blaNDM and blaKPC and one positive for blaNDM only). For SynMALDI, each isolate was incubated for 3 h in 4 tubes containing brain–heart infusion broth with the following: (1) no antibiotic; (2) ATM at 64 mg/L; (3) CZA at 10/4 mg/L; and (4) ATM at 64 mg/L plus CZA at 10/4 mg/L. After incubation, the bacterial protein extract was analyzed by MALDI-TOF MS, and the relative growth (RG) was determined for each isolate, considering intensities of the peaks of the bacterium incubated with antibiotic (tubes 2, 3, and 4) to the same bacterium incubated without antibiotic (tube 1), as follows: RG = IntensityWithantibiotic/IntensityWithoutantibiotic. The combination was determined as synergistic when there was an RG decrease of 0.3 in the antibiotic combination in relation to the RG of the most active antibiotic alone. Results: The combination of ATM plus CZA proved to be synergic by time–kill curve assay. All isolates tested with the SynMALDI method also presented synergism. Conclusions: Detection of synergism for ATM plus CZA combination can be determined by MALDI-TOF MS, providing fast results in order to improve patient treatment.

The modification of polymer chain ends is important in order to produce highly functional polymers. A novel chain-end modification of polymer iodides (Polymer-I) via reversible complexation-mediated polymerization (RCMP) with different functionalized radical generation agents, such as azo compounds and organic peroxides, was developed. This reaction was comprehensively studied for three different polymers, i.e., poly (methyl methacrylate), polystyrene and poly (n-butyl acrylate) (PBA), two different functional azo compounds with aliphatic alkyl and carboxy groups, three different functional diacyl peroxides with aliphatic alkyl, aromatic, and carboxy groups, and one peroxydicarbonate with an aliphatic alkyl group. The reaction mechanism was probed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The combination of PBA-I, iodine abstraction catalyst and different functional diacyl peroxides enabled higher chain-end modification to desired moieties from the diacyl peroxide. The dominant key factors for efficiency in this chain-end modification mechanism were the combination rate constant and the amount of radicals generated per unit of time.

Urinary tract infections (UTIs) are one of the most common human infections and are most often caused by Gram-negative bacteria such as Escherichia coli. In view of the increasing number of antibiotic-resistant isolates, rapidly initiating effective antibiotic therapy is essential. Therefore, a faster antibiotic susceptibility test (AST) is desirable. The MALDI-TOF MS-based phenotypic antibiotic susceptibility test (MALDI AST) has been used in blood culture diagnostics to rapidly detect antibiotic susceptibility. This study demonstrates for the first time that MALDI AST can be used to rapidly determine antibiotic susceptibility in UTIs directly from patients’ urine samples. MALDI-TOF MS enables the rapid identification and AST of Gram-negative UTIs within 4.5 h of receiving urine samples. Six urinary tract infection antibiotics, including ciprofloxacin, cotrimoxazole, fosfomycin, meropenem, cefuroxime, and nitrofurantoin, were analyzed and compared with conventional culture-based AST methods. A total of 105 urine samples from UTI patients contained bacterial isolates for MALDI AST. The combination of ID and AST by MALDI-TOF allowed us to interpret the result according to EUCAST guidelines. An overall agreement of 94.7% was found between MALDI AST and conventional AST for the urinary tract pathogens tested.

The potential development of a rapid and highly sensitive breast cancer (BC) diagnostic method has been increasingly investigated by many researchers in order to significantly improve the diagnosis of this disease that affects millions of women worldwide. Thus, this investigation aimed to establish a potential BC urinary peptidomic pattern using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a useful approach for BC diagnosis. The results of Lowry’s assay demonstrated that the total protein concentration increased after precipitation and that the healthy control group (HCs, 160 to 318 µg/mL, 142 ± DD µg/mL, on average) presented higher total protein content than the BC patients (140 to 311 µg/mL, 115 ± DD µg/mL, on average). Related to MALDI-TOF MS analysis, the results revealed that four peptide ion biosignatures (m/z 1046.5, 1062.5, 1237.7 and 1727.9) allowed the discrimination between BC patients and HCs. The distinction efficiency and accuracy of BC urine peptides were determined by receiver operating characteristic (ROC) curve analysis that enabled the recognition of some features with great sensitivity (88%) and specificity (98%). Therefore, the obtained data revealed MALDI-TOF MS as a powerful tool to explore peptidomic biosignatures due to its speed, sensitivity, and mass accuracy, which allow the establishment of novel disease biomarkers.

Francisella tularensis is a zoonotic bacterium that is endemic in large parts of the world. It is absent in the standard library of the most applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems: the Vitek MS and the Bruker Biotyper system. The additional Bruker MALDI Biotyper Security library contains F. tularensis without subspecies differentiation. The virulence of F. tularensis differs between the subspecies. The F. tularensis subspecies (ssp.) tularensis is highly pathogenic, whereas the subspecies holarctica displays lower virulence and subspecies novicida and F. tularensis ssp. mediasiatica are hardly virulent. To differentiate the Francisellaceae and the F. tularensis-subspecies, an in-house Francisella library was built with the Bruker Biotyper system and validated together with the existing Bruker databases. In addition, specific biomarkers were defined based on the main spectra of the Francisella strains supplemented with in silico genome data. Our in-house Francisella library accurately differentiates the F. tularensis subspecies and the other Francisellaceae. The biomarkers correctly differentiate the various species within the genus Francisella and the F. tularensis subspecies. These MALDI-TOF MS strategies can successfully be applied in a clinical laboratory setting as a fast and specific method to identify F. tularensis to subspecies level.

Francisella tularensis is a zoonotic bacterium that is endemic in large parts of the world. It is absent in the standard library of the most applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems: the Vitek MS and the Bruker Biotyper system. The additional Bruker MALDI Biotyper Security library contains F. tularensis without subspecies differentiation. The virulence of F. tularensis differs between the subspecies. The F. tularensis subspecies (ssp.) tularensis is highly pathogenic, whereas the subspecies holarctica displays lower virulence and subspecies novicida and F. tularensis ssp. mediasiatica are hardly virulent. To differentiate the Francisellaceae and the F. tularensis-subspecies, an in-house Francisella library was built with the Bruker Biotyper system and validated together with the existing Bruker databases. In addition, specific biomarkers were defined based on the main spectra of the Francisella strains supplemented with in silico genome data. Our in-house Francisella library accurately differentiates the F. tularensis subspecies and the other Francisellaceae. The biomarkers correctly differentiate the various species within the genus Francisella and the F. tularensis subspecies. These MALDI-TOF MS strategies can successfully be applied in a clinical laboratory setting as a fast and specific method to identify F. tularensis to subspecies level.

Detecting carbapenemase-associated carbapenem resistance is a subject of major clinical and epidemiological concern as it influences therapeutic choice. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a means to assess bacterial resistance mechanisms. We aimed to detect the KPC enzyme directly from positive blood cultures using MALDI-TOF MS. To do so, 102 clinical Enterobacteria were evaluated, including 59 blaKPC positives. Proteins were extracted using formic acid, isopropyl alcohol, and water (17:33:50) and spotted onto a steel target plate using the double-layer sinapinic acid technique. Two parameters were considered: (i) the visual detection of a clear peak with the expected KPC m/z and (ii) the evaluation of the relative intensity of the ions in the peak. A peak was observed in 56/59 blaKPC-positive isolates (94.9% sensitivity), with no false-positive results (100% specificity). When considering intensity, with a cut-off ≥120 (a.u.), sensitivity was 94.9% and specificity was 95.3%. We proposed a “buffer” zone, with intermediate values of intensity (115 to 125) reaching 100% sensitivity and specificity. The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid, which may improve appropriate patient therapy and antimicrobial stewardship.

International audience; Fleas are obligatory blood-sucking ectoparasites of medical and veterinary importance. The identification of fleas and associated flea-borne microorganisms, therefore, plays an important role in controlling and managing these vectors. Recently, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been reported as an innovative and effective approach to the identification of arthropods, including fleas. This study aims to use this technology to identify ethanol-preserved fleas collected in Vietnam and to use molecular biology to search for microorganisms associated with these fleas. A total of 502 fleas were collected from wild and domestic animals in four provinces in Vietnam. Morphological identification led to the recognition of five flea species, namely Xenopsylla cheopis, Xenopsylla astia, Pulex irritans, Ctenocephalides canis, and Ctenocephalides felis. The cephalothoraxes of 300 individual, randomly selected fleas were tested using MALDI-TOF MS and molecular analysis for the identification and detection of microorganisms. A total of 257/300 (85.7%) of the obtained spectra from the cephalothoraxes of each species were of good enough quality to be used for our analyses. Our laboratory MALDI-TOF MS reference database was upgraded with spectra achieved from five randomly selected fleas for every species of Ctenocephalides canis and Ctenocephalides felis. The remaining spectra were then queried against the upgraded MALDI-TOF MS database, which showed 100% correspondence between morphology and MALDI-TOF MS identification for two flea species (Ctenocephalides canis and Ctenocephalides felis). The MS spectra of the remaining species (three P. irritans, five X. astia, and two X. cheopis) were visually generated low-intensity MS profiles with high background noise that could not be used to update our database. Bartonella and Wolbachia spp. were detected in 300 fleas from Vietnam using PCR and sequencing with primers derived from the gltA gene for Bartonella and the 16S rRNA gene for Wolbachia, including 3 Bartonella clarridgeiae (1%), 3 Bartonella rochalimae (1%), 1 Bartonella coopersplainsensis (0.3%), and 174 Wolbachia spp. endosymbionts (58%).

Due to the global spread of pan resistant organisms, colistin is actually considered as one of the last resort antibiotics against MDR and XDR bacterial infections. The emergence of colistin resistant strains has been observed worldwide in Gram-negative bacteria, such as Enterobacteriaceae and especially in K. pneumoniae, in association with increased morbidity and mortality. This landscape implies the exploration of novel assays able to target colistin resistant strains rapidly.In this study, we developed and evaluated a new MALDI-TOF MS assay in positive-ion mode that allows quantitative or qualitative discrimination between colistin susceptible (18) or resistant (32) K. pneumoniae strains in 3 h by using the “Autof MS 1000” mass spectrometer. The proposed assay, if integrated in the diagnostic workflow, may be of help for the antimicrobial stewardship and the control of the spread of K. pneumoniae colistin resistant isolates in hospital settings.

Pigeon excreta can cause environmental and public health issues, particularly in urban and public areas. They are reservoirs of several human pathogens including fungi, bacteria, and viruses. Epidemiological data of pathogenic and opportunistic yeasts in pigeon droppings in Chon Buri, one of the most reputable tourist cities of Thailand, are scarce. The present study aimed to identify yeasts in pigeon droppings by MALDI-TOF mass spectrometry, and to study their prevalence in Chon Buri, Thailand. A total of 200 pigeon fecal samples were collected randomly from all 11 districts of Chon Buri. A sum of 393 yeast-like colonies were isolated on Sabourand’s dextrose agar and CHROMagar media. These isolates were further confirmed for their species by MALDI-TOF MS. Twenty-four yeast species belonging to 11 different genera were identified in pigeon fecal samples. Candida spp., predominantly C. krusei (14.32%), were the most prevalent yeast species. Other yeast species, including C. glabrata (12.73%), C. metapsilosis (11.93%), Lodderomyces elongisporus (10.87%), C. tropicalis (7.16%), C. albicans (5.83%), and Cryptococcus neoformans (4.77%) were identified. This study provides valuable epidemiological data and diversity of yeasts in pigeon droppings in Chon Buri, Thailand, and also supports the use of MALDI-TOF MS for yeast identification and epidemiological surveillance.

French Guiana mosquito fauna diversity encompasses 242 species, of which nearly half of them belong to Culex (Diptera: Culicidae) genus. Whereas several species from Culex genus are important vectors of arbovirus, scarce studies focus on Culex genus, notably due to the inaccurate morphological identification of field-caught females gender. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of mosquitoes. Culex females from French Guiana field were dissected based on morphological classification. Abdomens were used for molecular identification using COI (cytochrome oxydase 1) gene. Legs and thorax of 169 specimens belonging to 13 Culex species,( i.e. Cx. declarator, Cx. nigripalpus, Cx. quinquefasciatus, Cx. usquatus, Cx. adamesi, Cx. dunni, Cx. eastor, Cx. idottus, Cx. pedroi, Cx. phlogistus, Cx. portesi, Cx. rabanicolus and Cx. spissipes) were then submitted to MALDI-TOF MS analysis. A high intra-species reproducibility and inter-species specificity of MS spectra for each mosquito body part tested were noticed. A corroboration of the specimen identification was revealed between MALDI-TOF MS, morphological and molecular results. MALDI-TOF MS protein profiling proves to be a suitable tool for identification of neotropical Culex species and will permit to enhance knowledge on this highly diverse Culex mosquitoes genus.

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The N-15-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the N-15-labeling approach, the fates of supplemented N-15- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the N-15 atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (1,000 liquid samples daily at 1,000 liquid samples daily at

We report the first case of Coronavirus Disease 2019 (COVID-19)-associated brain abscess caused by a rare Trichosporon species, T. dohaense. The patient was a known diabetic and had received systemic corticosteroids for the treatment of COVID-19. He underwent craniotomy and evacuation of abscess. The pus aspirate grew a basidiomycetous yeast, morphologically resembling Trichosporon species. The isolate was initially misidentified by VITEK® MS due to lack of mass spectral database of T. dohaense. Accurate identification was achieved by internal transcribed spacer-directed panfungal polymerase chain reaction. The patient had a favorable outcome following surgical intervention and antifungal therapy.

Highly mucoid Streptococcus uberis were isolated on blood agar from five milk samples originating from two dairy cow farms. All the isolates were CAMP test and esculin hydrolysis negative. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) following prior extraction with 70% formic acid was used for identification to the species level. Given that it is plausible that such strains could be isolated in the future from dairy cows by laboratories in Serbia and the Balkans, we consider this case report to be a useful instruction for veterinary bacteriology laboratories.

International audience; Human infestations by bed bugs have upsurged globally in recent decades, including in African countries, where recent reports pointed out an increase in infestation. Sympatric dwelling has been described for two species of bed bug parasitizing humans: Cimex hemipterus (the tropical bed bug) and C. lectularius. Identification of these two species is based on morphological characteristics, and gene sequencing, and may also rely on Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS). The present work aimed to assess whether MALDI-TOF MS was applicable for species level identification of immature stages of Cimex. Arthropods were collected in domestic settings in Nouakchott, Mauritania. Identification used morphological keys and MALDI-TOF MS identification was assessed for immature stages. Quantitative PCR and sequencing assays were used to detect arthropod-associated bacteria in each specimen. A total of 92 arthropods were collected, all morphologically identified as C. hemipterus (32 males, 14 females and 45 immature stages). A total of 35/45 specimens produced good quality MALDI-TOF MS spectra. Analysis allowed species level identification of all immature C. hemipterus after their spectra were entered into our in-house MALDI-TOF MS arthropod spectra database. Molecular screening allowed detection of Wolbachia DNA in each specimen. These results suggested that MALDI-TOF MS is a reliable tool for species level identification of Cimex specimens, including immature specimens. Future studies should assess this approach on larger panels of immature specimens for different Cimex species and focus on the precise staging of their different immature developmental stages.

IntroductionMicrobial isolates from culture can be identified using 16S or whole-genome sequencing which generates substantial costs and requires time and expertise. Protein fingerprinting via Matrix-assisted Laser Desorption Ionization–time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid bacterial identification in routine diagnostics but shows a poor performance and resolution on commensal bacteria due to currently limited database entries. The aim of this study was to develop a MALDI-TOF MS plugin database (CLOSTRI-TOF) allowing for rapid identification of non-pathogenic human commensal gastrointestinal bacteria.MethodsWe constructed a database containing mass spectral profiles (MSP) from 142 bacterial strains representing 47 species and 21 genera within the class Clostridia. Each strain-specific MSP was constructed using >20 raw spectra measured on a microflex Biotyper system (Bruker-Daltonics) from two independent cultures.ResultsFor validation, we used 58 sequence-confirmed strains and the CLOSTRI-TOF database successfully identified 98 and 93% of the strains, respectively, in two independent laboratories. Next, we applied the database to 326 isolates from stool of healthy Swiss volunteers and identified 264 (82%) of all isolates (compared to 170 (52.1%) with the Bruker-Daltonics library alone), thus classifying 60% of the formerly unknown isolates.DiscussionWe describe a new open-source MSP database for fast and accurate identification of the Clostridia class from the human gut microbiota. CLOSTRI-TOF expands the number of species which can be rapidly identified by MALDI-TOF MS.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) är en viktig metod för artidentifiering av mikroorganismer på kliniska mikrobiologiska laboratorier. Enligt rekommendationer från Bruker Daltonics skall matrixapplicering ske relativt omedelbart efter att mikroorganismer har applicerats. Syftet med studien var att utvärdera effekter på artidentifiering vid förlängda tidsintervall mellan applicering av mikroorganismer och matrix med MALDI-TOF MS. Målet var att optimera arbetsflödet på mikrobiologilaboratoriet, laboratoriemedicin, Region Jönköping län (RJL). I studien undersöktes hur identifieringen av mikroorganismer påverkades av applicering avbakterie- och jästsvampstammar (n = 267) och α-Cyano-4-hydroxycinnaminsyra (HCCA)-matrix på en MALDI-provplatta vid varierande tidsintervall upp till tre timmar. Analys med MALDI-TOF MS genomfördes med masspektrometrarna MALDI Biotyper Sirius IVD System och Bruker MicroFlex LT. Resultaten visade att förlängda tidsintervall mellan applicering av mikroorganismer och matrix upp till tre timmar inte hade någon signifikant effekt på scorevärden vid artidentifiering, vilket innebar att tidsintervallen är acceptabla för artidentifiering med MALDI-TOF MS. Detta ger värdefull information för att optimera arbetsflödet och öka effektiviteten på mikrobiologilaboratoriet. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an important method for species level identification in clinical microbiology laboratories. According to recommendations from Bruker Daltonics matrix application should occur relatively immediately after microorganisms have been applied. The aim of the study was to evaluate the effects on microbial identification at extended time intervals between microbial and matrix application using MALDI-TOF MS. The objective was to optimize the workflow at the microbiology laboratory, laboratory medicine, Region Jönköping län (RJL). The study investigated the impact on identification of microorganisms between the application of bacterial and yeast strains (n = 267) and α-Cyano-4-hydroxycinnamic acid (HCCA) matrix on a MALDI target plate of various time intervals up to three hours. MALDI-TOF MS was performed using MALDI Biotyper Sirius IVD System and Bruker MicroFlex LT mass spectrometers.The results showed that a delay of matrix application up to three hours had no significant effect on score values, indicating that time did not affect the accuracy of microbial identification using MALDI-TOF MS. Consequently, the study provided valuable information for optimizing the workflow and increasing the efficiency of the microbiology laboratory.

Introduktion: Sepsis är utan behandling ett livshotande tillstånd, en korrekt behandling behöver en snabb och tillförlitlig artidentifiering. Olika prepareringsmetoder av positiva blododlingar inför direkt identifiering med MALDI-TOF finns. MBT Sepsityper® IVD Kit är en CE-märkt metod. För att följa IVDR-direktivet behöver Sepsityper jämföras med den nuvarande in-house-metoden. Dessa två metoder jämfördes utifrån förmåga att ge ett tillförlitligt, snabbt resultat där även den praktiska metoden samt ekonomiska aspekten undersöktes Material och metod: Alla positiva blododlingar som undersöktes preparerades med in- house-metoden och Sepsityper innan analys med MALDI-TOF MS. Med in-house-metoden användes saponin för att lysera de humana cellerna medans Sepsityper använde lyseringsbuffert. Resultatets tillförlitlighet presenteras som scorevärde där >2 är tillförlitligt till speciesnivå, 1,7–1,99 till genusnivå och, Background: Sepsis without treatment is a life-threatening condition, a rapid as well as reliable identification of the bacteria is needed. Different preparation methods of positive blood cultures for rapid identification are available. MBT Sepsityper® IVD Kit is one of them and is a CE-marked method. To comply with the IVDR directive, Sepsityper® needs to be tested and compared with the current in-house method. These two methods were compared based on the ability to provide fast and reliable results. Material and method: All the positive blood samples that were examined were prepared by the two methods then analysed with MALDI-TOF MS. The in-house-method used saponin to lyse the blood cells, Sepsityper used a lysisbuffert. How reliable the results are presented as score value, >2 means that the result is reliable to species identification, between 1.7-1.99 to genus identification and

Preservation of green palm leaves by silage preserves them as green feed in addition to the added value of probiotics and prebiotics. In this work, the characterization of fresh palm tree leaves was performed. The isolation of local lactic acid bacteria allowed selection of 4 isolates for the silage of the palm tree leaves. Dairy feed was used to enrich the leaves silage with the necessary nutrients. Lactobacillus paracasei and Pediococcus acidilactici were found to be dominating their respectively inoculated silages. The high occurrence of LAB found in the spontaneous silages of the palm tree leaves mixed with dairy feed is a good indication that ensiling palm tree leaves can support the growth of LAB and produce good-quality silage. However, a combination of the process by following the population dynamics using MALDI-TOF MS allowed the selection of the appropriate LAB strain, which is a new approach for application of silage. The publication of this article was funded by the Qatar National Library. Scopus

Acid-fast bacteria can be implicated in skin and soft tissue infections. Diagnostic identification can be challenging or not feasible by routine laboratory techniques, especially if there is no access to the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technology. Here, we present two cases of skin and soft tissue infections caused by two different acid-fast bacteria, Nocardia brasiliensis and Mycobacterium marinum. They both grew on Löwenstein–Jensen medium, Sabouraud agar medium and blood agar medium. Both bacteria appeared acid-fast by Ziehl–Neelsen stain and Gram-positive by Gram stain. The identification was performed by MALDI-TOF MS and gene analysis. N. brasiliensis and nontuberculous mycobacterium M. marinum represent rare pathogens that cause severe skin and soft tissue infections. Failure to identify the causative agent and subsequent inappropriate or inadequate treatment may lead to severe complications or even disseminated disease, especially in immunocompromised individuals.

The extensive use of tetracycline antibiotics has led to the widespread presence of tetracycline-resistance genes in Gram-negative bacteria and this poses serious threats to human and animal health. In our previous study, we reported a method for rapid detection of Tet(X)-producers using MALDI-TOF MS. However, there have been multiple machineries involved in tetracycline resistance including efflux pump, and ribosomal protection protein. Our previous demonstrated the limitation in probing the non-Tet(X)-producing tetracycline-resistant strains. In this regard, we further developed a MALDI-TOF MS method to detect and differentiate Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant strains. Test strains were incubated with tigecycline and oxytetracycline in separate tubes for 3 h and then analyzed spectral peaks of tigecycline, oxytetracycline, and their metabolite. Strains were distinguished using MS ratio for [metabolite/(metabolite+ tigecycline or oxytetracycline)]. Four control strains and 319 test strains were analyzed and the sensitivity was 98.90% and specificity was 98.34%. This was consistent with the results obtained from LC-MS/MS analysis. Interestingly, we also found that the reactive oxygen species (ROS) produced by tetracycline-susceptible strains were able to promote the degradation of oxytetracycline. Overall, the MALDITet(X)-plus test represents a rapid and reliable method to detect Tet(X)-producers, non-Tet(X)-producing tetracycline-resistant strains, and tetracycline-susceptible strains.

Actinomyces neuii can grow under aerobic culture conditions and shows a gram-positive rod morphology, similar to that of Corynebacterium spp. A. neuii is usually detected in local pus samples, and published cases of A. neuii bloodstream infections are rare. Here, we report a case of bloodstream infection caused by A. neuii subsp. anitratus. A 53-year-old woman with fever and hypotension was referred to our hospital. The patient underwent surgery for breast cancer and received chemotherapy after central venous (CV) port placement. On day 2, a blood culture in an anaerobic bottle yielded positive results, and A. neuii subsp. anitratus was identified via matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) and 16S rRNA sequencing. The patient was diagnosed with bloodstream infection caused by A. neuii subsp. anitratus with CV port infection. The CV port was removed and antibiotic treatment resulted in symptom improvement so the patient was discharged on day 28 of hospitalization. MALDI–TOF MS and 16S rRNA sequencing were found to be more useful for the identification of A. neuii than for phenotypic identification. Further research on A. neuii subsp. anitratus infections is required to avoid delayed or missed diagnoses.

The rapid identification of pathogens of bloodstream infections (BSIs) and the detection of antibiotic resistance markers are critically important for optimizing antibiotic therapy and infection control. The purpose of this study was to evaluate two approaches based on MALDI-TOF MS technology for direct identification of Gram-negative bacteria and automatic detection of Klebsiella pneumoniae carbapenemase (KPC) producers using the Bruker MBT Subtyping IVD Module in a large routine laboratory over a three-year period. MALDI-TOF MS analysis was performed directly from blood culture (BC) bottles following bacterial pellet recovery by Rapid MBT Sepsityper® Kit and on blood agar 4-h subcultures. Automated detection of blaKPC-carrying pKpQIL-plasmid by Bruker MBT Subtyping Module was evaluated in BCs tested positive to K. pneumoniae or E. coli. The results were compared with those obtained with conventional reference methods. Among the 2858 (93.4%) monomicrobial BCs, the overall species identification rates of the Rapid Sepsityper and the short-term subculture protocols were 84.5% (n = 2416) and 90.8% (n = 2595), respectively (p < 0.01). Excellent specificity for KPC-producers identification were observed for both MALDI-TOF MS protocols. The pKpQIL plasmid-related peak was detected in overall 91 of the 120 (75.8%) KPC-producing isolates. Notably, 14 out of the 17 (82.3%) K. pneumoniae isolates carrying blaKPC variants associated with ceftazidime/avibactam resistance and tested negative by the immunocromatography assay, were correctly identified as KPC-producers by MALDI-TOF MS. In conclusion, combination of both Rapid Sepsityper and short-term subculture protocols may represent an optimal solution to promptly identify more than 95% of Gram-negative bacteria causing BSIs. MALDI Biotyper® platform enabled a reliable and robust automated detection of KPC producers in parallel with species identification. However, integration of molecular or immunocromatographic assays are recommended according to local epidemiology.

One of the most common types of tinea is the superficial infection of the hair and scalp area known as tinea capitis. It is responsible for frequent outbreaks in nurseries and schools and represents a global health problem. Correct identification of the infection agent is essential in the determination of the infection source, epidemiological course, and treatment initiation. The conventional identification methods (direct exam, culture, DNA sequencing) are time-consuming, require experienced staff, are time-consuming, and the latter is expensive for routine identifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is gaining new ground for routine identification of filamentous fungi. The main advantages of MALDI-TOF MS are its rapid and accurate identification capability, relatively low cost, and easy integration into the laboratory routine. Its accuracy heavily depends on the quality of the reference spectra database. Identification of clinical isolates with MALDI-TOF MS protocol requires a sub-culturing step to ensure reliable identification. It can take days to weeks before fungal growth appears on solid medium. In this study, a unique MALDI-TOF MS protocol using liquid cultures of dermatophyte species was developed in order to shorten the turnaround time for the culture and identification of clinical isolates. Material and Method A standard MALDI-TOF MS protocol was adapted for liquid instead of solid cultures. Three different databases were tested. Results Using the liquid media MALDI-TOF MS protocol, a global rate of 62% correct identification (RCI) was obtained, compared with 87% for the protocol based on solid cultures. Trichophyton tonsurans was not correctly identified in all isolates using liquid cultures, with 88% of the isolates misidentified as Trichophyton interdigitale. The turnaround time for primary isolates for the solid and liquid protocols were respectively 11.7 and 11.6 days (no significant difference between both methods (p = 0.96)). Conclusions The newly designed liquid MALDI-TOF MS protocol did not lead to a significantly shorter turnaround time for the identification of dermatophytes isolated from tinea capitis infections. The turnaround time for the method with primary isolates was not significantly lower, and the rate of correct identification decreased remarkably, which emphasizes the need for a sub-culturing step. Using different database did not lead to improvement in turnaround time or rate of correct identification. This study highlights the importance of the medium and the reference database when performing MALDI-TOF MS.

Mangrove plants are rich in tannins, especially condensed tannins (CTs), which play an important role in biogeochemistry in coastal wetland ecosystem due to their functions of binding nutrients and heavy metal chelation. This study aims to obtain authentic chemical structures of mangrove CTs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Four organic solvents (n-hexane, ether, chloroform, and EtOAc (ethyl acetate)) were used for extraction tests and three purification methods (Method 1, Sephadex LH-20 absorbed tannins, and nontannins fraction were washed by 50% methanol (mp) solution; Method 2, Sephadex LH-20 absorbed tannins and nontannins fraction were washed by 100% ethanol (ep), and released in 70% acetone solution; and Method 3, Yb3+ selectivity precipitated tannins) were conducted to investigate their influences on the characterization of CTs from two mangrove species, Bruguiera gymnorhiza and Kandelia obovata. The results showed that (1) EtOAc was used as an extraction solvent, leading to unauthentic structural properties of CTs; (2) the distribution patterns of the polymers in mangrove CTs purified with 50% methanol elicited the least different trends with those of CTs in the two mangrove crude extracts, and the lower oligomers (dimmer-hexamer) and higher polymers were lost during purification of CTs by 100% ep. Therefore, based on the toxicity and price of solvents, the crude CTs from mangrove plants can be extracted with n-hexane or ether to remove lipid and pigment impurities and then purified with 50% methanol, which is a complete set of methods to obtain completely authentic structural information of mangrove CTs. This study can offer more accurate structural information of mangrove CTs and new insights for the conservation of mangrove living environments for follow-up research.

Multidrug-resistant (MDR) Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) has increased markedly in recent years, which has currently posed a major challenge in antimicrobial treatments and raised concerns regarding possible transfer of such bacteria through the food chain. The aim of this research was to investigate the prevalence of ESBL-producing E. coli in milk, cheese and meat samples and to determine their virulence, pathotype, serotype, antibiotic resistance and genetic relatedness. A total of 300 food samples were purchased from public markets in different districts of Giresun city. Five (1.6%) of 300 food samples resulted positive for ESBL-producing E. coli isolation. β-Lactamase-encoding genes of the CTX-M (20%), TEM (40%), and SHV (20%) groups were detected singly or in combination. Five ESBL-producing E. coli isolates were further analysed for the presence of virulence genes. Virulence factor genes detected were hlyA (20%), ehlyA (20%), iutA (60%), iucD (40%), fimH (100%), kpsMTII (100%) and traT (100%). Of 130 E.coli isolates, 5 ESBL-producing E. coli strains (totally 3.8%) were isolated, including 3 EHEC (60%). No EIEC, ETEC, EPEC, DAEC or EAggEC3 was detected. In PCR, three stx2 (60%) and one flicH7 (20%) encoding genes were found in ESBL-producing E. coli isolates. In addition, genes encoding Shiga toxins were detected in 3 of 5 isolates, three isolates (60%) encoded O128 serotype.The antibiotic susceptibility test of positive isolates showed resistant to cefuroxime, trimethoprim/ sulfamethoxazole, cefazolin, streptomycin, ceftriaxone, tetracycline, ampicillin and trimethoprim. Most of ESBL-producing E. coli isolates showed 80% MDR phenotypes against at least four classes of antibiotics. Specific-PCR detection of antibiotic resistance genes showed the prevalence of the tetA gene in most of the isolates (80%), followed by dfrA, qnr, aadA1 and sul1. PFGE results show that the isolates from different districts presented no clonal relatedness. This is the first report of the characteristics of multidrug resistance ESBL-producing shigatoxigenic E. coli in dairy and meat products in a local city in Turkey. Our findings indicate that dairy and meat products could be reservoirs of MDR ESBL-producing E. coli strains that were possessed several virulence factors and may be a cause of concern for human health.

MALDI-ToF MS spectra of protein extraction, digested by trypsin, from modern tissue samples (skin, fin, or muscle) of six different Pleuronectiformes species.&nbsp

International audience; Leishmania infantum is the agent of visceral leishmaniasis in the Mediterranean basin. It is transmitted by sandflies of the subgenus Larroussius. Although Phlebotomus perniciosus is the most important vector in this area, an atypical Ph. perniciosus easily confused with Ph. longicuspis has been observed in North Africa. MALDI-TOF MS, an important tool for vector identification, has recently been applied for the identification of sandflies. Spectral databases presented in the literature, however, include only a limited number of Larroussius species. Our objective was to create an in-house database to identify Mediterranean sandflies and to evaluate the ability of MALDI-TOF MS to discriminate close species or atypical forms within the Larroussius subgenus. Field-caught specimens (n = 94) were identified morphologically as typical Ph. perniciosus (PN; n = 55), atypical Ph. perniciosus (PNA; n = 9), Ph. longicuspis (n = 9), Ph. ariasi (n = 9), Ph. mascittii (n = 3), Ph. neglectus (n = 5), Ph. perfiliewi (n = 1), Ph. similis (n = 9) and Ph. papatasi (n = 2). Identifications were confirmed by sequencing of the mtDNA CytB region and sixteen specimens were included in the in-house database. Blind assessment on 73 specimens (representing 1073 good quality spectra) showed a good agreement (98.5%) between MALDI-TOF MS and molecular identification. Discrepancies concerned confusions between Ph. perfiliewi and Ph. perniciosus. Hierarchical clustering did not allow classification of PN and PNA. The use of machine learning, however, allowed discernment between PN and PNA and between the lcus and lcx haplotypes of Ph. longicuspis (accuracy: 0.8938 with partial-least-square regression and random forest models). MALDI-TOF MS is a promising tool for the rapid and accurate identification of field-caught sandflies. The use of machine learning could allow to discriminate similar species.

The effectiveness of differentiation of bacterial pathogens using MALDI-TOF mass spectrometry depends on the quality of sample preparation, compliance with mass spectrometric analysis parameters and statistical approaches used, implemented by various modern software tools. The review provides a brief description of the most known software used in the processing and bioinformation analysis of time-of-flight mass spectrometry data. A list of computer platforms, programs and environments, both commercial and publicly available, is presented. The results of indication and identification of pathogens of particularly dangerous and natural-focal infections by MALDI-TOF mass spectrometry using publicly available software – programming language R, Mass-Up, MicrobeMS, licensed – MatLab, ClinProTools, as well as free web applications, including, Speclust, Ribopeaksare provided. The data on usage of such well-known platforms as MALDI BioTyper, SARAMIS Vitek-MS and Andromas (Andromas SAS, France) for inter- and intra-specific differentiation of closely related species are presented. Results of identification and differentiation of microorganisms applying MALDI-TOF mass spectrometry based on detection of specific proteins for cross-comparison – biomarkers – are given. The analysis shows that the programming language R environment is one of the publicly available universal platforms with an optimal combination of algorithms for processing and interpreting of a large array of mass spectrometric data.

Background Mycobacterium marseillense is a new species of the Mycobacterium avium complex. There has been only a few human infections caused by M. marseillense worldwide. Case presentation We report a case of tenosynovitis caused by M. marseillense in an immunocompetent adult in Japan. The isolate was initially identified as M. intracellulare using commercial real time polymerase chain reaction assays and later identified as M. marseillense with sequencing of the the rpoB and hsp65 regions, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Conclusions This is the first case reporting on M. marseillense generating a positive result with commercial real time PCR assays targeting MAC. Human infections associated by M. marseillense might be underreported due to similarities with Mycobacterium intracellulare. To accurately identify M. marseillese, MALDI-TOF MS might provide a rapid and reliable method.

Background Neovasculogenesis is characteristic of herniated lumbar discs, in which extruded nucleus pulposus is prone to heme iron-induced cytotoxicity (increased oxidative stress causing ferroptosis). However, recent analyses of neovascularization are very complicated, and the mechanism of action is rarely reported. Methods Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) was performed to analyze human herniated and nonherniated nucleus pulposus. Then, the clinical relevance of the MALDI-TOF MS results and Pfirrmann classification of the degenerative nucleus pulposus were analyzed. To explore the mechanism, the heme-induced ferroptosis effect was evaluated at both the tissue and cell levels using high-resolution MALDI-TOF MS and molecular biology methods. Results The spectra revealed that hemoglobin (Hb) and heme signals were greatly increased, thus serving as predictors of vasculogenesis in herniated nucleus pulposus. The clinical relevance analysis demonstrated that the intensity of Hb and heme peaks was closely related to the Pfirrmann classification of degenerative nucleus pulposus. Mechanistically, increased heme catabolism and downregulation of glutathione peroxidase 4 (GPX4) levels were detected in herniated nucleus pulposus, reflecting iron-dependent cell death or ferroptosis. Iron levels was also increased in herniated nucleus pulposus compared with that in nonherniated nucleus pulposus. Furthermore, accuracy mass measurements confirmed that the levels of ferroptosis-related metabolites, such as glutathione, arachidonic acid (AA), sphinganine, polyunsaturated fatty acid (PUFA), and tricarboxylic acid (TCA) cycle metabolites, were significantly different between herniated and nonherniated tissues, indicating that the interior of the herniated tissues is a pro-oxidant environment. Moreover, heme-induced ferroptosis was verified in human nucleus pulposus cells (HNPCs), and the underlying mechanism might be associated with the Notch pathway. Conclusions Neovascularization in herniated nucleus pulposus may expose tissues to high levels of heme, which can induce cytotoxicity and ferroptosis within tissues and accelerate the progressive degeneration of herniated nucleus pulposus. This study is beneficial for understanding the pathological mechanism of herniated nucleus pulposus and facilitating the development of nonoperative interventions for treating lumbar disc herniation (LDH).

Infections with various bacteria, especially gram-negative aerobes, are a well-recognized problem in captive cold-blooded animals with immunocompromised health status, or in those kept under poor conditions. Pseudomonas is one of the most represented genera. Here, we present a case of fatal disseminated infection caused by Pseudomonas aeruginosa in a captive green iguana kept at the “Pionirska dolina” Zoo in Sarajevo, Bosnia and Herzegovina. At necropsy, severe stomatitis, pneumonia, hepatitis and nephritis, accompanied with focally extensive dermatitis were observed. Histopathology revealed multifocal necrosis in various visceral organs. Culture and subsequent MALDI-TOF MS analysis were conducted to identify the isolate as P. aeruginosa. Antimicrobial susceptibility testing revealed a wide susceptibility of the isolate, however applied therapy was instilled too late in the presented case. This case demonstrates the significance of timely and accurate identification, and antimicrobial susceptibility testing of bacterial isolates implicated in the pathology of captive reptiles. The importance of monitoring the adequate environmental conditions (enclosure temperature, humidity and conformation), health status and possible clinical signs of illness are highlighted.

Black porgy is an important marine aquaculture fish species whose production is at the fifth position in all kinds of marine-cultured fishes in China. In this study, Illumina high-throughput sequencing technology was used to sequence the total RNA of black porgy. Sixty-one candidate SNPs (Single Nucleotide Polymorphism) were screened out and genotyped through GATK4 (Genome Analysis ToolKit) software and MALDI-TOF MS (Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry). The experimental results showed that a total of sixty SNPs were successfully genotyped, with a success rate of 98.36%. The results of principal component analysis and correlation analysis of growth traits showed that body weight was the first principal component, with a cumulative contribution rate of 74%. There were significant correlations (p < 0.05) or extremely significant correlations (p < 0.01) between different growth traits. The results of genetic parameter analysis and association analysis showed that scaffold12-12716321, scaffold13-4787950, scaffold2-13687576 and scaffold290-11890 were four SNPs that met the requirement of polymorphic information content and conformed to the Hardy–Weinberg equilibrium. There were significant differences between their genotype and the phenotype of growth traits. The four SNP molecular markers developed in this research will lay a foundation for further exploration of molecular markers related to the growth traits of black porgy and will provide a scientific reference for the further study of its growth mechanisms. At the same time, these molecular markers can be applied to the production practices of black porgy, so as to realize selective breeding at the molecular level and speed up the breeding process.

In order to further accelerate pathogen identification from positive blood cultures (BC), various sample preparation protocols to identify bacteria with MALDI-TOF MS directly from positive BCs have been developed. We evaluated an in-house method in comparison to the Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) as well as the benefit of an on-plate formic acid extraction step following positive signal by the BACTECTM FX system. Confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 113 monomicrobial positive BCs were analyzed. The rates of Gram-positive bacteria correctly identified to the genus level using in-house method and Sepsityper® Kit were 63.3% (38/60) and 81.7% (49/60), respectively (p = 0.025). Identification rates at species level for Gram-positive bacteria with in-house method and Sepsityper® kit were 30.0% (18/60) and 66.7% (40/60), respectively (p < 0.001). Identification rates of Gram-negative bacteria were similar with the in-house method and Sepsityper® Kit. Additional on-plate formic acid extraction demonstrated significant improvement in the identification rate of Gram-positive bacteria at both genus and species level for both in-house (p = 0.001, p < 0.001) and Sepsityper® Kit methods (p = 0.007, p < 0.001). Our in-house method is a candidate for laboratory routines with Sepsityper® Kit as a back-up solution when identification of Gram-positive bacteria is unsuccessful.

Leishmania infantum is the agent of visceral leishmaniasis in the Mediterranean basin. It is transmitted by sandflies of the subgenus Larroussius. Although Phlebotomus perniciosus is the most important vector in this area, an atypical Ph. perniciosus easily confused with Ph. longicuspis has been observed in North Africa. MALDI-TOF MS, an important tool for vector identification, has recently been applied for the identification of sandflies. Spectral databases presented in the literature, however, include only a limited number of Larroussius species. Our objective was to create an in-house database to identify Mediterranean sandflies and to evaluate the ability of MALDI-TOF MS to discriminate close species or atypical forms within the Larroussius subgenus. Field-caught specimens (n = 94) were identified morphologically as typical Ph. perniciosus (PN; n = 55), atypical Ph. perniciosus (PNA; n = 9), Ph. longicuspis (n = 9), Ph. ariasi (n = 9), Ph. mascittii (n = 3), Ph. neglectus (n = 5), Ph. perfiliewi (n = 1), Ph. similis (n = 9) and Ph.papatasi (n = 2). Identifications were confirmed by sequencing of the mtDNA CytB region and sixteen specimens were included in the in-house database. Blind assessment on 73 specimens (representing 1073 good quality spectra) showed a good agreement (98.5%) between MALDI-TOF MS and molecular identification. Discrepancies concerned confusions between Ph. perfiliewi and Ph. perniciosus. Hierarchical clustering did not allow classification of PN and PNA. The use of machine learning, however, allowed discernment between PN and PNA and between the lcus and lcx haplotypes of Ph. longicuspis (accuracy: 0.8938 with partial-least-square regression and random forest models). MALDI-TOF MS is a promising tool for the rapid and accurate identification of field-caught sandflies. The use of machine learning could allow to discriminate similar species.

Peer reviewed: True, Applying MALDI-ToF mass spectrometry as a clinical diagnostic test for viruses is different from that of bacteria, fungi and other micro-organisms. This is because the systems biology of viral infections, the size and chemical nature of specific viral proteins and the mass spectrometry biophysics of how they are quantitated are fundamentally different. The analytical challenges to overcome when developing a clinical MALDI-ToF mass spectrometry tests for a virus, particularly human pathogenic enveloped viruses, are sample enrichment, virus envelope disruption, optimal matrix formulation, optimal MALDI ToF MS performance and optimal spectral data processing/bioinformatics. Primarily, the instrument operating settings have to be optimized to match the nature of the viral specific proteins, which are not compatible with setting established when testing for bacterial and many other micro-organisms. The capacity to be a viral infection clinical diagnostic instrument often stretches current mass spectrometers to their operational design limits. Finally, all the associated procedures, from sample collection to data analytics, for the technique have to meet the legal and operational requirement for often high-throughput clinical testing. Given the newness of the technology, clinical MALDI ToF mass spectrometry does not fit in with standard criteria applied by regulatory authorities whereby numeric outputs are compared directly to similar technology tests that have already been authorized for use. Thus, CLIA laboratory developed test (LDT) criteria have to be applied. This article details our experience of developing a SAR-CoV-2 MALDI-ToF MS test suitable for asymptomatic carrier infection population screening.

The understanding of the mechanism of Topo I inhibition by organic ligands is a crucial source of information that has led to the design of more effective and safe pharmaceuticals in oncological chemotherapy. The vast number of inhibitors that have been studied in this respect over the last decades have enabled the creation of a concept of an ‘interfacial inhibitor’, thereby describing the machinery of Topo I inhibition. The central module of action of this machinery is the interface of a Topo I/DNA/inhibitor ternary complex. Most of the ‘interfacial inhibitors’ are primarily kinetic inhibitors that form molecular complexes with an “on–off” rate timing; therefore, all of the contacts between the inhibitor and both the enzyme and the DNA are essential to keep the complex stable and reduce the “off rate”. To test this hypothesis, we designed the compound using a C-9-(N-(2′-hydroxyethyl)amino)methyl substituent in an SN38 core, with a view that a flexible substituent may bind inside the nick of a model of the DNA and stabilize the complex, leading to a reduction in the “off rate” of a ligand in a potential ternary complex in vivo. Using docking analysis and molecular dynamics, free energy calculations on the level of the MM-PBSA and MM-GBSA model, here we presented the in silico-calculated structure of a ternary complex involving the studied compound 1. This confirmed our suggestion that compound 1 is situated in a groove of the nicked DNA model in a few conformations. The number of hydrogen bonds between the components of a ternary complex was established, which strengthens the complex and supports our view. The docking analysis and free energy calculations for the receptor structures which were obtained in the MD simulations of the ternary complex 1/DNA/Topo I show that the binding constant is stronger than it was for similar complexes with TPT, CPT, and SN38, which are commonly considered as strong Topo I inhibitors. The binary complex structure 1/DNA was calculated and compared with the experimental results of a complex that was in a solution. The analysis of the cross-peaks in NOESY spectra allowed us to assign the dipolar interactions between the given protons in the calculated structures. A DOSY experiment in the solution confirmed the strong binding of a ligand in a binary complex, having a Ka of 746 mM−1, which was compared with a Ka of 3.78 mM−1 for TPT. The MALDI-ToF MS showed the presence of the biohybrid, thus evidencing the occurrence of DNA alkylation by compound 1. Because of it having a strong molecular complex, alkylation is the most efficient way to reduce the “on–off” timing as it acts as a tool that causes the cog to brake in a working gear, and this is this activity we want to highlight in our contribution. Finally, the Topo I inhibition test showed a lower IC50 of the studied compound than it did for CPT and SN38.

POCI01-0145-FEDER-007688 2021.03255.CEECIND Melanins play a fundamental role in the biology and ecology of several fungal species. Unfortunately, this group of amorphous macromolecules also severely (and most times irreversibly) stains cultural heritage objects. Despite efforts made throughout the years, knowledge of the chemical composition and structure of melanins is still insufficient, which hampers the task of safely cleaning these colourants from cultural heritage materials in a targeted way without causing further deterioration. This work aimed therefore to contribute towards enlightening the characteristics of fungal melanins from three fungi that are common paper colonizers: Aspergillus niger, Chaetomium globosum and Cladosporium cladosporioides. The extracted melanins were characterized by FTIR, Raman, UV-vis, Solid-State NMR and MALDI-TOF MS spectroscopies and the effect of inhibitors of DHN-melanin and DOPA-melanin pathways on colony pigmentation and growth was evaluated. Although all the extracted colourants show a predominantly aromatic structure with carbonyl and phenolic groups, some differences between the melanins can be highlighted. Melanins obtained from Ch. globosum and Cl. cladosporioides exhibited similar structures and composition and both presented DHN-melanin characteristics, while A. niger’s melanins revealed a more complex and ordered structure, with a higher prevalence of highly conjugated carbonyls than the others, besides the additional presence of a yellow/green component. These conclusions cannot be overlooked while selecting targeted cleaning methodologies for melanin stains on cultural heritage materials. publishersversion published

A symbiotic or mixed animal husbandry (e.g., pigs and chickens) is considered to have a positive effect for animal welfare and sustainable agriculture. On the other hand, a risk of infection and transmission of microorganisms, especially of zoonotic pathogens, between animal species may potentially occur and thus might increase the risk of foodborne illnesses for consumers. To prove these assumptions, two groups of animals and their environmental (soil) samples were investigated in this study. Animals were kept in a free-range system. In the first group, pigs and chickens were reared together (pasture 1), while the other group contained only pigs (pasture 2). During a one-year study, fecal swab samples of 240 pigs and 120 chickens, as well as 120 ground samples, were investigated for the presence of Campylobacter spp., Salmonella spp. and E. coli. Altogether, 438 E. coli and 201 Campylobacter spp. strains were isolated and identified by MALDI-TOF MS. Salmonella spp. was not isolated from any of the sample types. The prevalences of Campylobacter coli and C. jejuni in pigs were 26.7% and 3.3% in pasture 1 and 30.0% and 6.7% in pasture 2, while the prevalences of C. coli and C. jejuni in chickens from pasture 1 were 9.2% and 78.3%, respectively. No correlation between the rearing type (mixed vs. pigs alone) and the prevalence of Campylobacter spp. was observed. All swab samples were positive for E. coli, while the average prevalences in soil samples were 78.3% and 51.7% in pasture 1 and 2, respectively. Results of similarity analysis of the MALDI-TOF MS spectra (for C. coli, C. jejuni and E. coli) and FT-IR spectra (for E. coli) of the same bacterial species showed no recognizable correlations, no matter if strains were isolated from chickens, pig or soil samples or isolated at different sampling periods. The results of the study indicate that the symbiotic husbandry of pigs and chickens neither results in an increased risk of a transmission of Campylobacter spp. or E. coli, nor in a risk of bacterial alteration, as shown by MALDI-TOF MS and FT-IR spectra. In conclusion, the benefits of keeping pigs and chickens together are not diminished by the possible transmission of pathogens.

The prompt presumptive identification of methicillin-resistant Staphylococcus aureus (MRSA) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can aid in early clinical management and infection control during routine bacterial identification procedures. This study applied a machine learning approach to MALDI-TOF peaks for the presumptive identification of MRSA and compared the accuracy according to staphylococcal cassette chromosome mec (SCCmec) types. We analyzed 194 S. aureus clinical isolates to evaluate the machine learning-based identification system (AMRQuest software, v.2.1, ASTA: Suwon, Korea), which was constructed with 359 S. aureus clinical isolates for the learning dataset. This system showed a sensitivity of 91.8%, specificity of 83.3%, and accuracy of 87.6% in distinguishing MRSA. For SCCmec II and IVA types, common MRSA types in a hospital context, the accuracy was 95.4% and 96.1%, respectively, while for the SCCmec IV type, it was 21.4%. The accuracy was 90.9% for methicillin-susceptible S. aureus. This presumptive MRSA identification system may be helpful for the management of patients before the performance of routine antimicrobial resistance testing. Further optimization of the machine learning model with more datasets could help achieve rapid identification of MRSA with less effort in routine clinical procedures using MALDI-TOF MS as an identification method.

International audience; This study used MALDI-TOF MS and molecular tools to identify tick species infesting camels from Tamanrasset in southern Algeria and to investigate their associated microorganisms. Ninety-one adult ticks were collected from nine camels and were morphologically identified as Hyalomma spp., Hyalomma dromedarii, Hyalomma excavatum, Hyalomma impeltatum and Hyalomma anatolicum. Next, the legs of all ticks were subjected to MALDI-TOF MS, and 88/91 specimens provided good-quality MS spectra. Our homemade MALDI-TOF MS arthropod spectra database was then updated with the new MS spectra of 14 specimens of molecularly confirmed species in this study. The spectra of the remaining tick specimens not included in the MS database were queried against the upgraded database. All 74 specimens were correctly identified by MALDI-TOF MS, with logarithmic score values ranging from 1.701 to 2.507, with median and mean values of 2.199 and 2.172 ± 0.169, respectively. One H. impeltatum and one H. dromedarii (2/91; 2.20%) tested positive by qPCR for Coxiella burnetii, the agent of Q fever. We also report the first detection of an Anaplasma sp. close to A. platys in H. dromedarii in Algeria and a potentially new Ehrlichia sp. in H. impeltatum.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry has been widely used as an emerging technology for the rapid identification of microorganisms. Cronobacter sakazakii (C. sakazakii) is a food-borne pathogen of particular importance to the powdered infant formula (PIF) processing environment due to its high lethality in infants. However, the traditional solid spotting detection method of pretreating samples for MALDI-TOF MS leads only to qualitative detection of C. sakazakii. We developed a new, low-cost, robust liquid spotting pretreatment method and used a response surface methodology to optimize its parameters. The applicability, accuracy, and quantitative potential were measured for different types of samples. The optimal parameters of this method were as follows: a volume of 70% formic acid of 25 μL, treatment with ultrasound at 350 W for 3 min, and a volume of acetonitrile added of 75 μL. These conditions led to the highest identification score for C. sakazakii (1926.42 ± 48.497). This method was found to detect bacteria accurately and reproducibly. When 70 strains of C. sakazakii isolates were analyzed with this method, the identification accuracy was 100%. The detection limit of C. sakazakii in environmental and PIF samples was 4.1 × 101 cfu/mL and 2.72 × 103 cfu/mL, respectively.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is routinely used for bacterial identification. It would be highly beneficial to also be able to use the technology as a fast way to detect clinically relevant clones of bacterial species. However, studies to this aim have often had limited success. The methods used for data acquisition, processing and data interpretation are highly diverse amongst studies on MALDI-TOF MS sub-species typing. In addition to this, feasibility may depend on the bacterial species and strains investigated, making it difficult to determine what methods may or may not work. In our paper, we have reviewed recent research on MALDI-TOF MS typing of bacterial strains. Although we found a lot of variation amongst the methods used, there were approaches shared by multiple research groups. Multiple spectra of the same isolate were often combined before further analysis for strain distinction. Many groups used a protein extraction step to increase resolution in their MALDI-TOF MS results. Peaks at a high mass range were often excluded for data interpretation. Three groups have found ways to determine feasibility of MALDI-TOF MS typing for their set of strains at an early stage of their project.

Bovine mastitis is a costly disease in the dairy farms globally. The control of such disease is generally based on the prevention by the strict hygienic measures during milking. Other approaches include vaccination and the application of antibiotics. Regardless to these procedures, mastitis is not entirely under the control, thus increasing the need for alternative tactics. This study was conducted to isolate and identify lactic acid bacteria (LAB) from fresh cow’s milk which possess antibacterial activity that could be used for mastitis control. 146 isolates were recognized as (LAB) from105 milk tanks samples after being cultured anaerobically on de Man, Rogosa and Sharpe (MRS) agar plates for 48 hours at 37 oC and identified by general bacteriological investigation. Afterwards, 24 isolates were identified to belong to genus Lactobacillus using polymerase chain reaction (PCR), and for species level recognition MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was used resulted in : L. fermentum (5), L. brevis (3), L. plantarum (4), L. paracasei (2), L. rhamnosus (3), L. pentosus (2), L. casei (3), L. raffinolactis (1) and L. mesenteroids (1). The antimicrobial activity of these strains against one of the major mastitis pathogens, S. aureus, was detected by the agar well diffusion assay and the modified double layer method , where L. casei, L. fermentum and L. plantarum possess the most inhibiting effect besides they have no hemolytic nor gelatin liquefaction activity when their safety profiles were evaluated. The result of the antibiotic susceptibility test revealed that these isolates were resistant to vancomycin (VA), neomycin (N) and gentamycin (CN). On the other hands, they were highly sensitive to amoxicillin clavulanic acid (AMC), levamisole (LEV), tetracycline (TE) and penicillin (P). The study suggests that L. casei, L. fermentum and L. plantarum are perfect candidates to be used as probiotics to help in preventing and controlling bovine mastitis caused by Staphylococcus aureus as they were proven to be safe and have antimicrobial activity against the organism.

Corynebacterium (C.) diphtheriae is one of the two etiological pathogens for human diphtheria with significant morbidity and mortality. Recently, members of its biovar Belfanti have been described as two novel species, C. belfantii and C. rouxii. The most important virulence factor and also the premise to cause diphtheria is the isolate’s capacity to encode and express the diphtheria toxin (DT). In contrast to C. ulcerans, which represents a potentially zoonotic pathogen, C. diphtheriae (incl. the novel deduced species) has almost exclusively been found to comprise a human pathogen. We here report three rare cases of C. rouxii isolation from dogs suffering from disseminated poly-bacterial exsudative to purulent dermatitis and a traumatic labial defect, respectively. The isolates were identified as C. diphtheriae based on commercial biochemistry and matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI-TOF MS) analysis. However, recently described specific spectral peaks were highly similar to spectra of C. rouxii, which was confirmed by whole genome sequencing. Further investigations of the dog isolates for the presence of DT by tox gene qPCR revealed negative results. The findings from this study point out that skin infections in companion animals can be colonized by uncommon and so believed human specific pathogens, thereby resembling the clinical signs of cutaneous diphtheria.