Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In ...comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment — Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel
Congeria jalzici
, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches.
Acinetobacter species are common inhabitants of freshwater and marine ecosystems with a capacity to induce disease in affected fish. To facilitate their rapid and reliable identification, ...matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was applied to Acinetobacter from fish. The aims of the study were, thus, to identify and profile the Acinetobacter species from tissues of fish living in a vulnerable environment impacted by wastewaters, and to assess the potential of MALDI-TOF MS as a method for discriminating these acinetobacters. Fish were sampled from waterways impacted by the activity of a wastewater treatment plant. Samples of gills, spleen, kidney and liver were streaked onto general-purpose media to purity. The profiling and identification of acinetobacters was conducted with MALDI-TOF MS, with the samples prepared by ethanol/formic acid extraction.The identified acinetobacters were retrieved from gills (68.96 %), kidney (13.79 %), liver (10.34 %), and spleen (6.89 %). The Acinetobacter species isolated from all tested fish tissues were A. johnsonii (79.31 %), A. pittii (10.34 %), A. tandoii (3.44 %), A. guilouiae (3.44 %), and A. gerneri (3.44 %). Highly probable and probable species identifications were obtained for 48.27 % of all acinetobacters tested, indicating fully reliable identification. MALDI?TOF MS gave excellent identification and profiling results for piscine Acinetobacter species from the wastewater-affected waterways. It is a recommendable technique for future Acinetobacter species discrimination, as accurate and rapid identification of these bacteria is critical in both environmental pollution management and human/veterinary clinical diagnostics.
Pathogenic oomycete Aphanomyces astaci, the causative agent of crayfish plague, is listed among the world's 100 worst invasive alien species due to its fast spread and detrimental impact on native ...European crayfish populations, as well as losses in crayfish aquaculture. During the infection process, A. astaci mycelium invades the host through its cuticle where it interacts with the resident epibiotic bacteria. Since host microbial communities may have an important role in the defense against pathogens, in this study we aimed to i) identify the epibiotic bacterial isolates from the cuticle of two commercially relevant crayfish – the signal crayfish Pacifastacus leniusculus and the narrow-clawed crayfish Pontastacus leptodactylus, and ii) distinguish those that can inhibit the growth of A. astaci mycelium. Results of 16S rRNA gene sequencing and MALDI–TOF mass spectrometry revealed that the majority of collected isolates belonged to the phylum Proteobacteria (69%), followed by Bacteroidetes (17%) and Firmicutes (8%). Using the in vitro plate assays, we tested the potential of the isolates to inhibit A. astaci mycelial growth and classified them as inhibitors (33%) or non-inhibitors (67%). More than half of the identified inhibitors (56%) belonged to the genus Pseudomonas. Our results represent the first step in the understanding of interactions between A. astaci and epibiotic bacteria on the crayfish cuticle and open the perspective for testing in vivo the application of selected bacterial inoculums for crayfish plague prevention and/or treatment. The development of such A. astaci biocontrol measures is urgently needed in astaciculture.
•Bacteria were isolated from the cuticle of commercially relevant crayfish.•A third of the isolates inhibited the growth of crayfish pathogen A. astaci.•Pseudomonas isolates were the most potent A. astaci inhibitors.•Our results could start the development of new approaches in A. astaci control.
Vibrio species are widely distributed and can be potentially pathogenic to aquatic organisms. In this study, we isolated Vibrio spp. from environmental samples (seawater, sediment, and fish swabs) ...collected over a three-year period from a fish farm in Mali Ston Bay in the Adriatic Sea, Croatia, and assess their distribution. A total of 48 seawater samples and 12 sediment samples, as well as gill and skin swabs from 110 farmed European seabass, were analysed for the presence of Vibrio. Vibrio strains were identified to the species level by MALDI TOF MS. The analysis revealed that V. alginolyticus was the predominant species in European seabass, followed by V. anguillarum. V. alginolyticus was isolated from the sediments, along with V. gigantis and V. pomeroyi, while V. chagasii, V. cyclitrophicus, V. fortis, V. gigantis, V. harveyi, V. pelagius, and V. pomeroyi were isolated from seawater. V. anguillarum was isolated only twice during two different spring seasons, once from a diseased sea bass and the second time from a healthy sea bass. We analysed these two isolates and found that they differ both genetically and in terms of resistance to antibiotics. Our results confirm the seasonality of vibriosis incidence and the presence of the pathogenic V. anguillarum, which increases the risk of vibriosis.
Bacteria from the genus
Shewanella
are inhabitants of marine and freshwater ecosystems, recognized fish spoilage bacteria, but less known as fish disease agents.
Shewanella
spp. isolated from fish ...living in waters close to effluents of a wastewater treatment plant (WWTP) were not previously characterized. We have tested
Shewanella
isolates from WWTP-affected waters and related fish. Genotypic characterization identified most strains as
S. baltica
and
S. oneidensis
. In order to investigate the sensibility and accuracy of their MALDI-TOF MS identification, they were grown on two culture media enriched by various NaCl concentrations, incubated at different temperatures and duration. We analyzed their antimicrobial susceptibility on a panel of antimicrobial drugs and capacity for biofilm production. With a view to demonstrate their capacity to produce fatty acids, we assessed the impact of different culture media on their lipid profile. We performed zebrafish embryotoxicity tests to simulate the environmental infection of the earliest life stages in
S. baltica
-contaminated waters. The best MALDI-TOF MS identification scores were for strains cultivated on TSA for 24 h at 22 °C and with supplementation of 1.5% NaCl. Less than 17% of isolates demonstrated antimicrobial resistance. Most isolates were weak biofilm producers. Strain-to-strain variation of MIC and MBC was low. The major fatty acids were C15:0, C16:0, C16:1, C17:1, and iC15:0. Exposure of
Danio rerio
to different
S. baltica
concentrations induced severe effects on zebrafish development: decreased heartbeat rate, locomotor activity, and melanin pigmentation.
S. baltica
passed through chorionic pores of zebrafish.
The aim of this study was to compare the effectiveness of gas chromatography (GC) and near‐infrared spectroscopy (NIR) spectroscopy in detecting butter adulteration. Butter samples with a known ...percentage of added margarine or pork fat were prepared. Chemometric techniques (principal component analysis and partial least squares regression) were applied on the NIR spectra and GC data, which enabled the qualitative separation of samples depending on the added proportion of adulterant, and the quantitative prediction of the proportion of added adulterant or certain fatty acid content. C12:0; C16:0; C18:0; C16:1; C18:1n9; C18:2n6 showed significant trend changes depending on the adulteration degree. NIR has proven to be a suitable technique for the rapid determination of butter adulteration.
Novelty impact statement
Fast and nondestructive technique as near‐infra red (NIR) spectroscopy can be used in qualitative and quantitative detection of butter originality. Obtained models by chemometrics methods allow prediction of proportion of added adulterant in butter and identify specific fatty acids. NIR may be applied in screening control of the correctness of butter labeling to improve the system of quality control in food fraud.
The novel N‐1‐sulfonylcytosine‐cyclam conjugates 1 and 2 conjugates are ionized by electrospray ionization mass spectrometry (ESI MS) in positive and negative modes (ES+ and ES−) as singly ...protonated/deprotonated species or as singly or doubly charged metal complexes. Their structure and fragmentation behavior is examined by collision induced experiments. It was observed that the structure of the conjugate dictated the mode of the ionization: 1 was analyzed in ES− mode while 2 in positive mode. Complexation with metal ions did not have the influence on the ionization mode. Zn2+ and Cu2+ complexes with ligand 1 followed the similar fragmentation pattern in negative ionization mode.
The transformation from 2°‐amine in 1 to 3°‐amine of cyclam ring in 2 leads to the different fragmentation patterns due to the modification of the protonation priority which changed the fragmentation channels within the conjugate itself. Cu2+ ions formed complexes practically immediately, and the priority had the cyclam portion of the ligand 2. The structure of the formed Zn2+ complexes with ligand 2 depended on the number of 3° amines within the cyclam portion of the conjugate and the ratio of the metal:ligand used. The cleavage of the cyclam ring of metal complexes is driven by the formation of the fragment that suited the coordinating demand of the metal ions and the collision energy applied. Finally, it was shown that the structure of the cyclam conjugate dictates the fragmentation reactions and not the metal ions.
Acinetobacter baumannii is an emerging hospital pathogen. Whereas A. baumannii isolated from patients or hospitals has been reported, there are few data regarding propagation of viable A. baumannii ...in the natural environment. This study investigates the occurrence and antimicrobial susceptibility of viable A. baumannii in municipal wastewater and its persistence through the wastewater treatment process. A total of 21 A. baumannii isolates were recovered at a secondary type of municipal wastewater treatment plant in Zagreb, Croatia: 15 from raw influent wastewater and six from final effluent. All isolates were carbapenem- and multidrug-resistant. Among 14 isolates tested for blaOXA genes, all harboured the constitutive blaOXA-51-like gene, while the acquired blaOXA-23-like and blaOXA-40-like genes were found in 10 and three isolates respectively. Six A. baumannii isolates recovered from effluent wastewater multiplied and survived in sterilised effluent wastewater up to 50 days. These findings support the idea that multidrug-resistant A. baumannii can occur and have the ability to survive in the environment.
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