In this study, the purposefulness of using the API20E biochemical identification system as a supportive tool for enhancing the discrimination of environmental bacteria by MALDI-TOF MS method was ...evaluated. The identification results of MALDI-TOF MS and API20E for 321 Gram-negative strains isolated from the riverine freshwater and its sediment, and from the tissues of fish from the same water body were compared. Of 190 isolates identified with probable to highly probable species-level identification, and secure genus to probable species identification, 14 isolates (7.37%) had identification score over 2.300, and from the same group 19 isolates (10%) had excellent or very good identification to the genus by API20E system. With regard to agreement at genus level, out of 231 strains with genus designation available by API20E at any level of identification reliability, MALDI-TOF MS genus identification agreed in 163 (70.6%) strains. Of these, 135 (82.8%) were Aeromonas species and the remaining isolates belonged to 7 different genera. Although API20E resulted in frequent misidentification due to a limited profile index, its individual biochemical reactions might contribute to overall characterization of isolates. For example, for all reliable A. hydrophila strain identifications with MALDI-TOF MS, ONPG, GLU and OX reactions were unarguably positive for all fish and water/sediment isolates, whereas only fish isolates yielded additional 100% positive TDA and VP reactions. Thus, after initial identification with MALDI-TOF MS, environmental isolates with lower identification scores should be further analyzed. Before commencing confirmatory testing with nucleic acid-based methods, biochemical API20E tests could be applied as a purposeful and inexpensive identification support in targeting better identification accuracy. In this study, this was particularly evident with A. hydrophila, Chryseobacterium sp. and Pseudomonas sp. This identification strategy could significantly resolve methodological and cost-related shortcomings frequently occurring with large number of environmental isolates.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific ...applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI‐TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI‐TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements.
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•As-rich mixture of pharmacolite, hornesite, and talmessite from Crven Dol mine.•Stenotrophomonas sp., Microbacterium spp. and bacterial consortium were isolated.•Novel ...hyper-resistant bacteria survive 32 g/L arsenite and 176 g/L arsenate.•As-tolerance involve efflux systems including ArsB, Acr3(1) and Acr3(2) proteins.•SEM/EDS analyses confirmed intracellular accumulation of arsenic.
Novel hyper-resistant bacteria were isolated from the Crven Dol mine (Allchar, North Macedonia), arsenic-rich extreme environment. Bacteria were recovered from a secondary mineral mixture, an alteration of hydrothermal realgar rich in arsenates (pharmacolite, hornesite, and talmessite). The sample was recovered from the dark part of the mine at 28 m depth. Three bacterial strains and a bacterial consortium were isolated for their capacity to survive exposure to 32 g/L (209 mM) of arsenite, and 176 g/L (564 mM) of arsenate. The 16S rRNA gene analysis identified bacterial isolates as Stenotrophomonas sp. and two Microbacterium spp. This analysis also revealed that bacterial consortium comprise two Bacteriodetes exhibiting similarity to Olivibacter ginsengisoli and to uncultured bacterium, and one γ-proteobacteria with similarity to Luteimonas sp. Among all isolates Stenotrophomonas sp. exhibited the highest tolerance to As compound as well as the capacity to accumulate As inside the cells. Analysis of genes involved in As-resistance showed that recovered isolates possess the genes encoding the ArsB, Acr3(1) and Acr3(2) proteins, indicating that at least a part of their resistance could be ascribed to As-efflux systems described in isolates obtained from human-polluted environments.
Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid ...identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48-72 hours.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gallibacterium anatis is considered one of the most common bacterial causative agents of reproductive tract disorders in poultry. In this study, phylogenetic analysis of partial rpoB sequences and ...biotyping using MALDI-TOF MS was done in order to investigate the genetic diversity of Gallibacterium isolates from 13 farms with different biosecurity measures and management practices. Sampling was done as a part of regular monitoring, except for Farms 9-13 that were included in the study to represent extensive production systems with lowest biosecurity levels. Pharyngeal and cloacal swabs were taken from live birds, while swabs from trachea, liver, peritoneum and oviduct were taken during necropsies. After cultivation and identification, strains from each farm were randomly selected for sequencing and biotyping. Both results showed high level of heterogeneity among the isolates originating from farms with low biosecurity levels, unlike isolates from farms with higher biosecurity levels and proper management that were more closely related and clustered together. Such correlation was statistically significant. Low biosecurity levels enable horizontal transmission of the pathogens, as well as gene transfer. The results confirm the importance of adequate biosecurity measures and management on poultry farms as they greatly affect the genetic diversity of the pathogens. Therefore, implementation of basic biosecurity measures could help control the heterogeneity of Gallibacterium strains, which would alleviate control of the infection prevalence on farms through immunoprophylaxis, and consequently improve poultry production. Also, the genetic diversity of G. anatis on poultry farms could be a good bioindicator of management practices and biosecurity measures used.
RESEARCH HIGHLIGHTS
High correlation between low biosecurity and high diversity of Gallibacterium anatis.
Diversity of Gallibacterium is a good bioindicator of management practices on farms.
Human milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life but also contains a variety of bacteria that play a key role in tailoring ...the neonatal faecal microbiome. Microbiome analysis of human milk and infant faeces from mother-breastfed infant pairs was performed by sequencing the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq platform. According to the results, there is a connection in the composition of the microbiome in each mother-breastfed infant pair, supporting the hypothesis that the infant's gut is colonised with bacteria from human milk. MiSeq sequencing also revealed high biodiversity of the human milk microbiome and the infant faecal microbiome, whose composition changes during lactation and infant development, respectively. A total of 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) electrophoresis profiles of 100 strains isolated from human milk and identified by 16S RNA sequencing. Since certain cellular molecules may support their use as probiotics, the next focus was to detect (S)-layer proteins, bacteriocins and exopolysaccharides (EPSs) that have potential as therapeutic biomolecules. SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) coupled with LC-MS (liquid chromatography-mass spectrometry) analysis revealed that four
strains expressed S-layer proteins, which were identified for the first time in strains isolated from human milk. The potential biosynthesis of plantaricin was detected in six
strains by PCR analysis and
antibacterial studies.
H NMR (Proton Nuclear Magnetic Resonance) analysis confirmed EPS production in only one strain,
MC1. The overall microbiome analysis suggests that human milk contributes to the establishment of the intestinal microbiota of infants. In addition, it is a promising source of novel
strains expressing specific functional biomolecules.
•Improved protocol for the separation of glycolipids: SQDG, MGDG and DGDG.•Marine glycolipids are successfully separated and quantified by the new protocol.•New approach extends the power of TLC–FID ...enabling analysis of 18 lipid classes.
We demonstrate improved power of Iatroscan thin layer chromatography/flame ionization detection (TLC–FID) technique for analysis of complex marine lipid mixture by developing protocol for the separation and analysis of glycolipids including sulfoquinovosyldiacylglycerols (SQDG), monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG). We have modified the common protocol used so far for the analysis of lipid classes by replacing the elution step which uses pure acetone for the elution of acetone mobile polar lipids, with the elution step containing chloroform–acetone (72:28, v:v) for separation of MGDG and DGDG. To separate SQDG from the complex lipid matrix we introduced solvent mixture acetone–chloroform–methanol–formic acid (33:33:33:0.6, v:v:v:v). Quantification of glycolipid classes was performed after calibration with glycolipid standards for the masses between 0.2 and 2.7–5.0μg. With this new protocol we have successfully separated three glycolipids from the complex particulate lipid mixture of the seawater samples. Such an approach extends the power of existing protocol for the analysis of lipids which altogether ensure detection and quantification of 18 lipid classes what was demonstrated on seawater samples. This enables to gain a very broad system overview of the particularly complex environments as are seas, oceans and freshwaters.
(phylum
, class
) is a zoophilic opportunistic pathogen with recognized potential for invasive infections in humans. Although this pathogenic yeast is widespread in nature, it has been primarily ...studied in domestic animals, so available data on its genotypes in the wild are limited. In this study, 80 yeast isolates recovered from 42 brown bears (
) were identified as
by a culture-based approach. MALDI-TOF mass spectrometry (MS) was used to endorse conventional identification. The majority of samples exhibited a high score fluctuation, with 42.5% of isolates generating the best scores in the range confident only for genus identification. However, the use of young biomass significantly improved the identification of
at the species confidence level (98.8%). Importantly, the same MALDI-TOF MS efficiency would be achieved regardless of colony age if the cut-off value was lowered to ≥1.7. Genotyping of LSU, ITS1, CHS2, and β-tubulin markers identified four distinct genotypes in
.
isolates. The most prevalent among them was the genotype previously found in dogs, indicating its transmission potential and adaptation to distantly related hosts. The other three genotypes are described for the first time in this study. However, only one of the genotypes consisted of all four loci with bear-specific sequences, indicating the formation of a strain specifically adapted to brown bears. Finally, we evaluated the specificity of the spectral profiles of the detected genotypes. MALDI-TOF MS exhibited great potential to detect subtle differences between all
isolates and revealed distinct spectral profiles of bear-specific genotypes.
Two main parameters that structure the marine ecosystem by affecting species distribution, abundance, community structure, timing of major life cycle events, and trophic state of the ecosystem are ...temperature and nutrient availability. Faced with climate change, eukaryotic plankton cope at multiple levels through physiological accommodation, adaptive evolution, shift in time and/or space of habitat, and/or community responses. Thirty‐two years of our phytoplankton research indicate that Chaetoceros curvisetus/pseudocurvisetus adjust to climate change more successfully than the majority of the accompanying phytoplankton taxa in the mesotrophic part of the NW Adriatic Sea, the Mediterranean. While the abundance of the entire accompanying phytoplankton community has decreased significantly since 2003 (the period of the northern Adriatic warming and oligotrophication) compared to the previous period (1986–2003), the abundance of C. curvisetus/pseudocurvisetus remained unchanged, while its contribution to the community increased significantly. Accommodation strategies include a change in the timing of high abundance and blooms in the surface layer and successful blooming in the deeper layers during warm months. Apart from the observed in situ accommodation, physiological acclimation to warming may involve changes in photosynthesis, respiration, growth, and cell biochemistry. Here, we conducted laboratory experiments with C. pseudocurvisetus to investigate how warming affects its biochemical response through the fatty acid remodeling of phospholipid classes. Long‐term field observations and short‐term laboratory experiments suggest that marine diatoms C. curvisetus/pseudocurvisetus are potential global winners with the ability to acclimate/adapt to climate change.
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