MALDI-TOF MS Mass Spectrometry Research Articles

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

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

Oral colonization of Acinetobacter baumannii in intensive care units: Risk factors, incidence, molecular epidemiology, association with the occur of pneumonia and sepsis, and infection control measures.

Objective(s):Oral colonization of Acinetobacter baumannii can lead to infections such as pneumonia and sepsis. We aimed to evaluate oral colonization of hospitalized patients in ICUs and to examine risk factors for oral colonization, molecular epidemiology, and incidence of pneumonia and sepsis. Materials and Methods:The study began in February 2021. Oral cultures were taken. The microorganisms were identified by a Maldi-tof MS mass spectrometry device. Colistin resistance genes were investigated by polymerase chain reaction. Clonal relationships were determined by pulsed-field gel electrophoresis. Results: A. baumannii was found in 21 of 96 patients’ oral cultures. Pneumonia and sepsis due to A. baumannii were detected in 14 and 5 patients, respectively. The mean growth time of A. baumannii from oral cultures was 11.8 days, and the meantime for the occurrence of pneumonia after oral growth was 5.2 days. We determined a plasmid mediated mcr-2 colistin resistance gene in a colistin susceptible A. baumannii strain. It is the first report of the plasmid mediated mcr-2 colistin resistance gene in our country. In total, fourteen different A. baumannii genotypes were determined in PFGE. It was determined that the effects of antibiotic use, oral motor dysfunction, mechanical ventilation, intubation, orogastric tube use, and total parenteral nutrition intake on oral colonization were statistically significant.Conclusion:Oral colonization of A. baumannii is a significant concern in ICUs. We believe that it is important to take oral cultures and follow the risk factors and take infection control measures to prevent oral colonization of resistant isolates in ICUs.

In Vitro Antitumor Activity of Endophytic and Rhizosphere Gram-Positive Bacteria from Ibervillea sonorae (S. Watson) Greene against L5178Y-R Lymphoma Cells.

Plant-associated microorganisms represent a potential source of new antitumor compounds. The aim of the present study was to isolate endophytic and rhizosphere Gram-positive bacteria from Ibervillea sonorae and produce extracts with antitumor activity. Methanol and ethyl acetate extracts were obtained from 28 d bacterial fermentation, after which murine L5178Y-R lymphoma cells growth inhibition was evaluated at concentrations ranging from 15.62 µg/mL to 500 µg/mL by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide reduction colorimetric assay. IC50 and the selectivity index (SI) were calculated and compared with healthy control human peripheral blood mononuclear cells (PBMC). Identification of the isolated strains was performed using the 16S ribosomal gene and by MALDI-TOF MS mass spectrometry. The endophytic and rhizosphere bacterial extracts from strains ISE-B22, ISE-B26, ISE-B27, ISS-A01, ISS-A06, and ISS-A16 showed significant (p < 0.05) L5178Y-R cell growth inhibition, compared with an untreated control. The rhizosphere Micromonospora echinospora isolate ISS-A16 showed the highest (90.48%) percentage of lymphoma cells growth inhibition and SI (19.1) for PBMC, whereas the Bacillus subtilis ISE-B26 isolate caused significant (p < 0.01) growth inhibition (84.32%) and a SI of 5.2. Taken together, results of the present study evidenced antitumor effects by I. sonorae endophytic and rhizosphere bacteria culture extracts. Further research will involve the elucidation of the compounds that exert the antitumor activity and their evaluation in pre-clinical studies.

Study on the clinical application of Streptococcus pneumoniae serotype detection based on MALDI-TOF MS technology

Streptococcus pneumoniae is related to the prognosis of infected patients. If the strain can be identified to the serotype level, it will play a key role in formulating measures to prevent Streptococcus pneumoniae infection. Therefore, this study focuses on analyzing the significance of serotype detection of Streptococcus pneumoniae under the guidance of MALDI-TOF MS technology, and provides a reference for rapid clinical typing of Streptococcus pneumoniae. A total of 500 patients were selected for treatment in our hospital from April 2019 to April 2020. The main types of diseases were patients with sinusitis, pneumonia, bacteremia, and acute otitis media. A total of 500 cases were collected, and specimens of Streptococcus pneumoniae were collected. Observe the distribution of MALDI-TOF MS mass spectrometry scores; the composition of the serotype distribution of Streptococcus pneumoniae isolates; the molecular typing of Streptococcus pneumoniae isolates; the relationship between the clinical characteristics of 500 patients and the distribution of serotypes 14, 19F; the distribution of serotypes 14, 19F and Analysis of the relationship between patient prognosis. Capsular swelling, latex agglutination test, MALDI-TOF MS detection of serotype distribution of serotype Streptococcus pneumoniae isolates, serotypes 6B, 14, 19A, 19F, 23F accounted for a large proportion, of which the highest serotype was mainly 14. 19F; The number of serotypes detected by MALDI-TOF MS was the highest, reaching 489 (97.80%), the number of serotypes detected by capsular swelling was 308 (61.60%), and the number of serotypes detected by latex agglutination test was 313 (62.40%). The distribution of serotypes detected by MALDI-TOF MS was significantly higher than the other two detection techniques (P < 0.05), and there was no statistical difference between the two tests of capsular swelling and latex agglutination (P > 0.05). The distribution rates of serotypes 14 and 19F were compared in different ages, disease types, drugs, etc., and there were statistical differences (P < 0.05), but there was no statistical difference in gender (P > 0.05). The prognosis of 500 patients, of which 487 cases improved (serotype 14: 98 strains; serotype 19F: 100 strains), 13 died (serotype 14: 12 strains; serotype 19F: 12 strains), serotypes 14, 19F There is a significant correlation between the distribution and the prognosis of patients (P < 0.05). MALDI-TOF MS technology can quickly, accurately and simply identify Streptococcus pneumoniae, with high sensitivity and specificity, and can save time for clinical diagnosis of Streptococcus pneumoniae infection. The laboratory can use MALDI-TOF MS to collect protein profiles of known typed strains and construct a corresponding typing mass spectrum library, and perform principal component cluster analysis on the spectrum to be tested and the library, which can quickly type Streptococcus pneumoniae.

Actinotignum schaalii: reporte de dos casos de bacteriemias en Argentina

Actinotignum schaalii is an emerging pathogen in elderly patients with urinary tract pathologies. Two cases of A. schaalii bacteremia are described. Case 1: 79-year-old patient with a history of benign prostatic hyperplasia. He was admitted to the ward for febrile syndrome, abdominal pain, and dysuria. Case 2: 95-year-old patient with prostatic adenomectomy, urethrostomy due to urethral stricture, and benign prostatic hyperplasia. He was admitted due to febrile syndrome, productive cough, bilateral infiltrates with right paracardiac image, and pleural effusion. In both patients, A. schaalii was isolated in blood cultures, identified by MALDITOF-MS mass spectrometry. Only in case 1 was it confirmed that the focus of bacteremia was urinary. It is important to suspect this emerging pathogen in urinary infections with pathological sediment without developing in traditional culture media to ensure adequate empirical treatment. Since most of the isolates show resistance to ciprofloxacin and trimethoprim-sulfamethoxazole.

Homology analysis and clinical infection characteristics of hypervirulent Klebsiella pneumonia

Objective: The hvKP phenotype strains were screened from the sensitive and multi-drug resistant Klebsiella pneumoniae, and the distribution and homology of their clinical infection characteristics were compared. Methods: A total of 158 Klebsiella pneumoniae strains isolated from clinical infection specimens in the Third Affiliated Hospital of Sun Yat-sen University from 2014 to 2018 were retrospectively collected. The string test (ST) was used to screen the hvKP infection, and a total of 65 were screened. The number of patients included 51 males and 14 females, with an average age of 56 years. The composition ratio of Klebsiella pneumoniae was analyzed for clinical infection related information. Matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) was used to identify the hypervirulent Klebsiella pneumoniae and establish a new database, and bio-tree cluster analysis and principal component analysis were carried out for all strains that met the experimental conditions by using analysis software and the homology of protein level was obtained. Results: A total of 65 strains hvKP were screened, 62 of which were susceptible strains and 3 were multi-drug resistant strains. The main diseases of infected patients were diagnosed as hepatobiliary disease, encephalopathy and liver abscess. The basic diseases of the population were mainly diabetes and hypertension. Dendrogram cluster analysis divided hvKP into 3 groups at a distance of 550 from the horizontal line; principal component analysis found that some strains were closely related, and the Kc group with the largest proportion was mainly related to hepatobiliary diseases. Ka group was mainly related to infection after surgery. Conclusions: HvKP was highly invasive and the clinical manifestations of infection were diverse. The infected patients were mostly elderly patients with weakened immunity. The rapid homology analysis of MALDI-TOF MS mass spectrometry has important clinical significance for the rapid screening of hvKP infection.

Identification of Fungi by Conventional Microscopy Combined with Novel MALDI-TOF MS Mass Spectrometry

Identification of Fungi by Conventional Microscopy Combined with Novel MALDI-TOF MS Mass Spectrometry

The in vitro efficacy of eye drops containing a bacteriophage solution specific for Staphylococcus spp. isolated from dogs with bacterial conjunctivitis

BackgroundThe purpose of the study was to evaluate the in vitro antibacterial effect of experimental eye drops with bacteriophages in elimination of Staphylococcus spp. isolated from dogs with bacterial conjunctivitis.. The bacterial material was collected from dogs with independent clinical signs of bacterial conjunctivitis. Staphylococcus spp. were identified by phenotypic and genotypic methods (MALDI-TOF MS mass spectrometry). Antibiotic resistance was determined by the disc-diffusion method. Phage activity (Plaque forming units, PFU) was determined on double-layer agar plates. Phages with lytic titres > 108 PFU were used to prepare eye drops. The stability of the antibacterial titre was evaluated for preparations stored in sealed bottles as well as after opening and reclosing.ResultsThe tests confirmed the occurrence of Staphylococcus spp. strains as etiological agents of bacterial conjunctivitis in dogs. A high percentage of strains were resistant to more than three antibiotics. The experimental phage eye drops used in the study exhibited 100% efficacy in vitro against the tested Staphylococcus isolates. Particularly noteworthy is the long duration of activity and constant antibacterial lytic titre of ≥108 PFU/mL of two eye drop solutions, nos. 7 and 12, after the bottle had been opened (21 days) and after hermetically sealed packaging (28 days) at 4–8 °C.ConclusionsThe results represent the first stage of research and require continuation in vivo. If positive effects are obtained in animals, the results can be used in applied research in humans and animals.

Evaluation of MALDI-TOF MS for the identification of opportunistic pathogenic yeasts of clinical samples

MALDI-TOF MS mass spectrometry is a rapid and straightforward technique to identify microorganisms by protein analysis. The study was performed in 304 yeast isolates from superficial and deep mycoses, in order to compare three methods: conventional (biochemical and morphological), MALDI-TOF MS, and polymerase chain reaction (PCR, reference). We included 24 species with predominance of Candida spp and Cryptococcus spp. The identification by conventional methods was 258/304 strains, while by MALDI-TOF MS was: 277/304 strains (84.8% versus 91.2%, P = not significant). The Kappa coefficient comparing MALDI-TOF-MS with PCR reported excellent concordance (0.99). The sensitivity and specificity of MALDI-TOF MS for the diagnosis of opportunistic pathogenic yeasts of clinical samples were 94.6% and 99% respectively. MALDI-TOF MS is a simple, fast and reliable tool for pathogenic yeasts.

Antibacterial and antibiofilm activity of the bacteriocin-producing strain Leuconostoc mesenteroides CHBY46 isolated from Algerian dromedary milk against Pseudomonas aeruginosa and Staphylococcus aureus biofilms

In order to control biofilm formation of pathogenic and spoilage bacteria in foods, some species of Leuconostoc are very important in food industries, as they increase the shelf life of foods during preservation. In this study the strain CHBY46 a bacteriocin-producing strain belonging to Leuconostoc ge-nus isolated from dromedary milk in the south of Algeria was characterized by 16S rRNA gene sequencing and MALDITOF MS mass spectrometry, tested for its antibacterial and antibiofilm activities against Pseudomonas aeruginosa and Staphylococcus aureus. The produced bacteriocin was partially puri-fied with sulfate ammonium precipitation and RP-HPLC. The strain CHBY46 was classified as Leuc. mesenteroides after molecular identification. Among the bacteria tested the pathogens Staph. aureus ATCC 29213 and Ps. aeru-ginosa ATCC 27653 were sensitive to this bacteriocin with 480 AU/ml. Antibi-ofilm activity was investigated by crystal violet assay. The bacteriocin of Leuc. mesenteroides CHBY 46 exhibited significant biofilm inhibition ; 35.58% with Ps. aeruginosa, and 42.11% with Staph. aureus. Tricine-SDS-PAGE analysis of the partially purified bacteriocin indicated a low molecular weight of approximately 3.5 kDa. Therfore, we conclude that bacteriocins from Leuco-nostoc have the potential as a therapeutic strategy against pathogen’s bio-films, which contribute, to bacterial pathogenicity and resistance toward antibiotics or being used in foods as adjunsts to contribute food safety.

Control of carbapenem-resistant Acinetobacter baumannii outbreak in an intensive care unit of a teaching hospital in Southern Italy.

Background Acinetobacter baumannii is an opportunistic pathogen that has become a major cause of concern, since it is a frequent cause of healthcare-associated infections (HAIs). The aim of the study was to describe the occurrence, the management and the control of an outbreak that occurred in an intensive care unit (ICU) of a teaching hospital in Southern Italy caused by multiple strains of extensively drug-resistant A. baumannii (XDRAB).MethodsCase-patient was defined as a patient with an healthcare-associated infection caused by an XDRAB isolate identified in a clinically significant culture. Environmental samples were collected from different surfaces. The isolates were identified by typical Gram stain morphology, using the Vitek 2 system (bioMérieux, France) and by MALDI-TOF MS mass spectrometry (bioMèrieux, France). Genotyping was performed through rep-PCR analysis.ResultsA patient presented an XDRAB ventilator-associated pneumonia at admission and was managed with strict isolation precautions until discharge. Five patients had a ventilator-associated pneumonia and two had a central line-associated bloodstream infection. Of the environmental samples, 1 sample obtained from the side of the bed of an infected patient yielded growth of XDRAB. Infection control measures were adopted. Rep-PCR analysis identified four patterns.ConclusionsThe integration of epidemiological and microbiological data and the application of infection control measures were crucial to bring such an outbreak to a rapid halt. The distinctive characteristic of this study was the complex molecular pattern of the outbreak, which subsided in a short period of time due to adherence to infection-control measures, confirming the fundamental role of molecular typing in the comprehension of outbreaks dynamics and of integrated control interventions for the interruption of epidemic events.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-2036-7) contains supplementary material, which is available to authorized users.

Escherichia coli strains from ostriches and their sensitivity to antimicrobial substances.

Ostriches are bred especially for their high-quality meat. There is a lack of knowledge concerning the ostrich's microflora. Escherichia coli is a commensal microorganism of the poultry intestine, ostriches included. However, some strains may become pathogenic. This study was therefore undertaken to detect coliform bacteria in ostrich faeces and to test their antibiotic profile and sensitivity to enterocins. Faeces (n=54, 18 mixture samples from 3 different age groups of 140 ostriches) were sampled to isolate coliform bacteria. The counts of coliform bacteria varied from 5.69 ± 2.4 log10 CFU/g to 5.73 ± 2.4 CFU/g. Pure colonies were identified using MALDI-TOF MS mass spectrometry and confirmed by phenotypization. Seventy-one strains were allotted to the species E. coli. Sixty-four of those 71 strains caused hemolysis. They were mostly polyresistant to antibiotics. Thirty-two poly-resistant strains of E. coli were sensitive to enterocins. These strains were most sensitive to Ent 9296 (26 strains). Moreover, Ent EM41 produced by E. faecium EM41 (isolated from ostrich faeces) inhibited the growth of 20 strains, reaching activity of 100 AU/ml. Our results indicate the possibility of enterocins being used for prevention/reduction of coliforms. Of course, in vivo studies are also being processed.

Gram-Stain Plus MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) for a Rapid Diagnosis of Urinary Tract Infection

Microbiological confirmation of a urinary tract infection (UTI) takes 24–48 h. In the meantime, patients are usually given empirical antibiotics, sometimes inappropriately. We assessed the feasibility of sequentially performing a Gram stain and MALDI-TOF MS mass spectrometry (MS) on urine samples to anticipate clinically useful information. In May-June 2012, we randomly selected 1000 urine samples from patients with suspected UTI. All were Gram stained and those yielding bacteria of a single morphotype were processed for MALDI-TOF MS. Our sequential algorithm was correlated with the standard semiquantitative urine culture result as follows: Match, the information provided was anticipative of culture result; Minor error, the information provided was partially anticipative of culture result; Major error, the information provided was incorrect, potentially leading to inappropriate changes in antimicrobial therapy. A positive culture was obtained in 242/1000 samples. The Gram stain revealed a single morphotype in 207 samples, which were subjected to MALDI-TOF MS. The diagnostic performance of the Gram stain was: sensitivity (Se) 81.3%, specificity (Sp) 93.2%, positive predictive value (PPV) 81.3%, negative predictive value (NPV) 93.2%, positive likelihood ratio (+LR) 11.91, negative likelihood ratio (−LR) 0.20 and accuracy 90.0% while that of MALDI-TOF MS was: Se 79.2%, Sp 73.5, +LR 2.99, −LR 0.28 and accuracy 78.3%. The use of both techniques provided information anticipative of the culture result in 82.7% of cases, information with minor errors in 13.4% and information with major errors in 3.9%. Results were available within 1 h. Our serial algorithm provided information that was consistent or showed minor errors for 96.1% of urine samples from patients with suspected UTI. The clinical impacts of this rapid UTI diagnosis strategy need to be assessed through indicators of adequacy of treatment such as a reduced time to appropriate empirical treatment or earlier withdrawal of unnecessary antibiotics.

MALDI-TOF MS of Trichoderma: a model system for the identification of microfungi

This investigation aimed to assess whether MALDI-TOF MS analysis of the proteome could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of peptide mass fingerprints would reveal apomorphies that could be useful in diagnosing species in this genus. One hundred and twenty nine morphologically and genetically well-characterized strains of Hypocrea and Trichoderma, belonging to 25 species in 8 phylogenetic clades, were analyzed by MALDI-TOF MS mass spectrometry. The resulting peak lists of individual samples were submitted to single-linkage cluster analysis to produce a taxonomic tree and were compared to ITS and tef1 sequences from GenBank. SuperSpectra™ for the 13 most relevant species of Trichoderma were computed. The results confirmed roughly previously defined clades and sections. With the exceptions of T. saturnisporum (Longibrachiatum Clade) and T. harzianum (Harzianum Clade), strains of individual species clustered very closely. T. polysporum clustered distantly from all other groups. The MALDI-TOF MS analysis accurately reflected the phylogenetic classification reported in recent publications, and, in most cases, strains identified by DNA sequence analysis clustered together by MALDI-TOF MS. The resolution of MALDI-TOF MS, as performed here, was roughly equivalent to ITS rDNA. The MALDI-TOF MS technique analyzes peptides and represents a rough equivalent to sequencing, making this method a useful adjunct for determination of species limits. It also allows simple, reliable, and quick species identification, thus representing a valid alternative to gene sequencing for species diagnosis of Trichoderma and other fungal taxa.