In Vitro Evaluation of Synergistic Essential Oils Combination for Enhanced Antifungal Activity against Candida spp.

In recent years, a significant number of infections have been attributed to non-albicidal Candida species (NAC), mainly due to the increasing resistance of NAC to antifungal agents. As only a few antifungal agents are available (azoles, echinocandins, polyenes, allylamines and nucleoside analogues), it is very important to look for possible alternatives to inhibit resistant fungi. One possibility could be essential oils (EOs), which have been shown to have significant antifungal and antibacterial activity. Therefore, in this study, the efficacy of 12 EOs and their combinations was evaluated against four yeasts of the genus Candida (C. albicas, C. glabrata, C. tropicalis and C. parapsilosis). GC-MS and GC-MS FID techniques were used for the chemical analysis of all EOs. VITEK-2XL was used to determine the antifungal susceptibility of the tested Candida spp. strains. The agar disc diffusion method was used for primary screening of the efficacy of the tested EOs. The broth dilution method was used to determine the minimum inhibitory concentrations (MICs) of the most potent EOs. After MIC cultivation, the minimum fungicidal concentration (MFC) was determined on Petri dishes (60 mm). The synergistic effect of combined EOs was evaluated using the checkerboard method and expressed as a fractional inhibitory concentration index (FICI). The results showed that ginger > ho-sho > absinth > dill > fennel > star anise > and cardamom were the most effective EOs. For all Candida species tested, the synergy was mainly observed in these combinations: ginger/fennel for C. albicans FICI 0.25 and C. glabrata, C. tropicalis and C. parapsilosis FICI 0.5 and absinth/fennel for C. albicans FICI 0.3125, C. tropicalis FICI 0.3125 and C. parapsilosis FICI 0.375. Our results suggest that the resistance of fungal pathogens to available antifungals could be reduced by combining appropriate EOs.

Seasonal assessment of selected trace elements in grass carp (Ctenopharyngodon idella) blood and their effects on the biochemistry and oxidative stress markers.

Environmental pollution by anthropogenic activity is still a highly relevant global problem. Aquatic animals are a specifically endangered group of organisms due to their continuous direct contact with the contaminated environment. Concentrations of selected trace elements in the grass carp (Ctenopharyngodon idella) (n = 36) blood serum/clot were monitored. Possible effects of the elements on selected biochemical and oxidative markers were evaluated. The concentrations of trace elements (Al, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Ga, Mn, Mo, Ni, Pb, Sr, Tl, and Zn) were analysed in the fish blood serum and blood clot by inductively coupled plasma optical emission spectrometry (ICP OES). A general scheme of decreasing concentrations of trace elements in the blood serum samples was: Zn ˃ Fe ˃ Sr ˃ Ba ˃ Ni ˃ Al ˃ Cu ˃ Be ˃ Co; < LOQ (below limit of quantification): Bi, Cd, Cr, Ga, Mn, Mo, Pb, Tl; and in the case of the blood clot, the scheme was as follows: Fe ˃ Zn ˃ Sr ˃ Al ˃ Ni ˃ Ba ˃ Cu ˃ Be ˃ Co ˃ Mn; < LOQ (below limit of quantification): Bi, Cd, Cr, Ga, Mo, Pb, Tl. Significant differences among the seasons were detected. The Spearman R correlation coefficients and linear or non-linear regression were used to evaluate direct relationships between trace elements and selected blood biomarkers. The correlation analysis between biochemical parameters (Na, K, P, Mg, AST, ALT, ALP, GGT, TAG, TP, urea, glucose) and trace elements (Al, Ba, Be, Cu, Fe, Ni, Sr, and Zn) concentrations confirmed statistically significant interactions in both seasons (summer and autumn). The regression analysis between oxidative stress markers (ROS, GPx, creatinine, uric acid, and bilirubin) and elements (Al, Ba, Co, Cu, Fe, Ni, and Sr) content confirmed statistically significant interactions. The results point to numerous connections between the observed elements and the physiological parameters of freshwater fish.

Streptomyces iakyrus TA 36 as First-Reported Source of Quinone Antibiotic γ-Rubromycin.

A wide range of bioactive compounds with potential medical applications are produced by members of the genus Streptomyces. A new actinomycete producer of the antibiotic γ-rubromycin, designated TA 36, was isolated from an alpine soil sample collected in Peru (Machu Picchu). Morphological, physiological and biochemical characteristics of the strain, together with data obtained via phylogenetic analysis and MALDI-TOF MS, were used for the correct identification of the isolate. The isolate TA 36 showed morphological characteristics that were consistent with its classification within the genus Streptomyces. Phylogenetic analysis based on 16S rRNA gene sequences showed that the TA 36 strain was most similar to S. iakyrus and S. violaceochromogenes with 99% similarity. Phylogenetic analysis together with the profile of whole cell proteins indicated that the strain tested could be identified as S. iakyrus TA 36. The crude extract Ext.5333.TA 36 showed various effects against the tested organisms with strong antimicrobial activity in the growth of Staphylococcus aureus (Newman) (MIC value of 0.00195 µg/µL). HPLC fractionation and LC/MS analysis of the crude extract led to the identification of the quinone antibiotic γ-rubromycin, a promising antitumour and antibacterial antibiotic. To the best of our knowledge, there is currently no report on the production of γ-rubromycin by S. iakyrus. Therefore, this study suggests S. iakyrus TA 36 as the first-reported source of this unique bioactive secondary metabolite.

Pathogens in milk of goats and their relationship with somatic cell count.

We evaluated the presence of bacterial pathogens in the milk of goats and their relationship with somatic cell count (SCC) and milk composition. The study was performed on a dairy farm in northern Slovakia. Half udder milk samples were collected from goats in June and July. The samples were divided on the basis of SCC into 4 bands (SCC1 lowest to SCC4 highest). Bacterial pathogens were only detected in 13% of samples. SCC3 and SCC4 had 15 and 25% positive samples respectively compared with SCC1 (2%) and SCC2 (14%). Coagulase-negative staphylococci (CNS) were the most common isolates (73%), of which Staphylococcus caprae was the most frequently isolated (65%). In samples with ≥ 1000 × 103 cells ml- 1 (SCC3, SCC4) there was higher somatic cell score (SCS) in the presence of a pathogen (7.48 ± 0.11) than without a pathogen (7.16 ± 0.05, P < 0.01). Statistically significant but weak negative correlations were observed between SCS and lactose, dry matter and non-fat dry matter. In conclusion, a higher percentage of bacteriologically positive milk samples was observed in both SCC3 and SCC4 groups but this does not explain the aetiology of high SCC in the milk of goats that are apparently free of bacteria. As a diagnostic tool, SCC is probably less useful in goats than in cows.

MALDI-TOF MS Indirect Beta-Lactamase Detection in Ampicillin-Resistant Haemophilus influenzae.

Rapid identification of beta-lactamase-producing strains of Haemophilus influenzae plays key role in diagnostics in clinical microbiology. Therefore, the aim of this study was the rapid determination of beta-lactamase's presence in H. influenzae isolates via indirect detection of degradation ampicillin products using MALDI-TOF MS. H. influenzae isolates were subjected to antibiotic resistance testing using disk diffusion and MIC methodologies. Beta-lactamase activity was tested using MALDI-TOF MS, and results were compared to spectral analysis of alkaline hydrolysis. Resistant and susceptible strains of H. influenzae were distinguished, and strains with a high MIC level were identified as beta-lactamase-producing. Results indicate that MALDI-TOF mass spectrometry is also suitable for the rapid identification of beta-lactamase-producing H. influenzae. This observation and confirmation can accelerate identification of beta-lactamase strains of H. influenzae in clinical microbiology, which can have an impact on health in general.

Use of MALDI-TOF MS to Discriminate between Aflatoxin B1-Producing and Non-Producing Strains of Aspergillus flavus.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins. One of the producers of AFB1 is Aspergillus flavus. Therefore, its rapid identification plays a key role in various sectors of the food and feed industry. MALDI-TOF mass spectrometry is one of the fastest and most accurate methods today. Therefore, the aim of this research was to develop the rapid identification of producing and non-producing strains of A. flavus based on the entire mass spectrum. To accomplish the main goal a different confirmatory MALDI-TOF MS and TLC procedures such as direct AFB1 identification by scraping from TLC plates, A. flavus mycelium, nutrient media around A. flavus growth, and finally direct AFB1 identification from infected wheat and barley grains had to be conducted. In this experiment, MALDI-TOF mass spectrometry with various modifications was the main supporting technology. All confirmatory methods confirmed the presence of AFB1 in the samples of aflatoxin-producing strains of A. flavus and vice versa; AFB1 was not detected in the case of non-producing strains. Entire mass spectra (from 2 to 20 kDa) of aflatoxin-producing and non-producing A. flavus strains were collected, statistically analyzed and clustered. An in-depth analysis of the obtained entire mass spectra showed differences between AFB1-producing and non-producing strains of A. flavus. Statistical and cluster analysis divided AFB1-producing and non-producing strains of A. flavus into two monasteries. The results indicate that it is possible to distinguish between AFB1 producers and non-producers by comparing the entire mass spectra using MALDI-TOF MS. Finally, we demonstrated that if there are established local AFB1-producing and non-producing strains of A. flavus, the entire mass spectrum database identification of aflatoxigenic A. flavus strains can be even faster and cheaper, without the need to identify the toxin itself.

The Vapor Phase of Selected Essential Oils and Their Antifungal Activity In Vitro and In Situ against Penicillium commune, a Common Contaminant of Cheese.

This study aimed to determine the in vitro and in situ antifungal activity of (14) selected essential oils (EOS), namely clove, thyme, red thyme, litsea, eucalyptus, niaouli, fennel, anise, cumin, basil, rosemary, sage, bergamot mint, and marjoram, by vapor contact against the growth of two strains of Penicillium commune (KMi-183 and KMi-402). Furthermore, to exclude the negative effect of EOs on the lactic acid bacteria (LABs) (Streptococcus spp.) on cheeses, their influence was monitored. Next, the sensory evaluation of cheese treated by EOs was evaluated. The results show that litsea and clove EOs were the most effective in the vapor phase against both tested strains. These EOs were characterized by the highest amount of α- (40.00%) and ß-Citral (34.35%) in litsea and eugenol (85.23%) in clove. The antitoxicogenic activity of less effective (in growth inhibition) EOs on cyclopiazonic acid (CPA) production by the tested strains was also observed. The growth of Streptococcus spp. (ranging from 8.11 to 9.69 log CFU/g) was not affected by the EOs in treated cheese. Even though the evaluators recognized some EOs in sensory evaluation by the triangle test, they did not have a negative effect on the taste and smell of the treated cheeses and were evaluated as edible. The antifungal activity of EOs against several types of microscopic fungi and their effect on the sensory properties of treated foods needs to be further tested to achieve the most effective protection of foods from their direct contaminants.

Carbapenemase Producing Klebsiella pneumoniae (KPC): What Is the Best MALDI-TOF MS Detection Method.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria is a group of highly dangerous antibiotic resistant Gram-negative Enterobacteriaceae. They cause infections associated with significant morbidity and mortality. Therefore, the rapid detection of KPC-producing bacteria plays a key role in clinical microbiology. Matrix assisted laser desorption/ionization time-of- flight (MALDI-TOF) is a rapidly evolving technology that finds application in various clinical, scientific, and industrial disciplines. In the present study, we demonstrated three different procedures of carbapenemase-producing K. pneumoniae (KPC) detection. The most basic model of MALDI-TOF instrument MS Microflex LT was used, operating in the linear ion-positive mode, commonly used in modern clinical laboratories. The first procedure was based on indirect monitoring of carbapenemase production with direct detection of hydrolyzed carbapenem antibiotic degradation products in the mass spectrum. The second procedure was based on direct detection of blaKPC accompanying peak with an 11,109 Da in the mass spectrum of carbapenemase-producing K. pneumoniae (KPC), which represents the cleaved protein (pKpQIL_p019) expressed by pKpQIL plasmid. In addition, several unique peaks were detected in the carbapenemase-producing K. pneumoniae (KPC) mass spectrum. The third procedure was the identification of carbapenemase-producing K. pneumoniae (KPC) based on the protein fingerprint using local database created from the whole mass spectra. By comparing detection procedures, we determined that the third procedure was very fast and relatively easy. However, it requires previous verification of carbapenemase-producing K. pneumoniae (KPC) using other methods as genetic blaKPC identification, detection of carbapenem degradation products, and accompanying peak with 11,109 Da, which represents cleaved pKpQIL_p019 protein expressed by pKpQIL plasmid. Detection of carbapenemase-producing K. pneumoniae using MALDI-TOF provides fast and accurate results that may help to reduce morbidity and mortality in hospital setting when applied in diagnostic situations.

Antifungal and Antitoxigenic Effects of Selected Essential Oils in Vapors on Green Coffee Beans with Impact on Consumer Acceptability.

The main objective of this study is to evaluate the effect of selected essential oils thyme chemotype linalool (Thymus zygis L.), thyme chemotype tymol (Thymus vulgaris L.), eucalyptus (Eucalyptus globulus Labill.), lavender (Lavandula angustifolia Mill.), mint (Mentha piperita L.), almond (Prunbus dulcis Mill.), cinnamon bark (Cinnamomum zeylanicum Nees), litsea (Litsea cubeba Lour. Pers), lemongrass (Cympogon citrati L. Stapf), and ginger (Zingiber officinalis Rosc.) in the vapor phase on growth, sporulation, and mycotoxins production of two Aspergillus strains (Aspergillus parasiticus CGC34 and Aspergillus ochraceus CGC87), important postharvest pathogens of green and roasted coffee beans. Moreover, the effect of the essential oils (EOs) on the sensory profile of the coffee samples treated with EOs was evaluated. The major components of tested EOs were determined by gas chromatography and mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). The results showed that almond, cinnamon bark, lemongrass, and litsea EOs are able to significantly inhibit the growth, sporulation, and mycotoxins production by toxigenic fungi. Sensory evaluation of coffee beans treated with EOs before and after roasting showed that some EOs (except lemongrass and litsea) do not adversely affect the taste and aroma of coffee beverages. Thus, application of the vapors of almond and cinnamon EOs appears to be an effective way that could serve to protect coffee during its transport and storage from toxigenic fungi.

Antifungal and synergistic activities of some selected essential oils on the growth of significant indoor fungi of the genus Aspergillus.

The study aimed to assess the antifungal activity of twenty-five essential oils (EOs) and the potential synergistic activity of the most effective EOs against significant indoor fungi of the genus Aspergillus [A. fumigatus (KBio-122), A. flavus (KBio-134), A. terreus (KBio-145) and A. niger (KBio-202)]. The chemical composition of all EOs was evaluated by the gas chromatography coupled with mass spectrometry (GC/MS) and gas chromatography with flame ionization detector (GC-FID) analysis. The antifungal susceptibility of EOs was evaluated by using the broth microdilution method. The most effective EOs were selected to determine the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) at a concentration range from 256 to 0.125 µg/mL. For the synergistic activities, the most effective EOs were tested using the chessboard pattern. The most sensitive strain to treatments with essential oils alone and in the combination of EOs was A. flavus (KBio-134). The chessboard assay showed that combinations of lemongrass and thyme EOs proved the most potent synergistic antifungal activity (FICI = 0.1875) against A. fumigatus (KBio-122). The synergy displayed by a combination of some EOs may be used to control fungal growth or increasing resistance to available synthetic antifungals, consequently permitting the reduction of their most active doses.

Diagnostic Methods of Clostridioides difficile Infection and Clostridioides difficile Ribotypes in Studied Sample.

BACKGROUND: Clostridioides (Clostridium) difficile is the most common nosocomial pathogen and antibiotic-related diarrhea in health-care facilities. Over the last few years, there was an increase in the incidence rate of C. difficile infection cases in Slovakia. In this study, the phenotypic (toxigenicity, antimicrobial susceptibility) and genotypic (PCR ribotypes, genes for binary toxins) patterns of C. difficile isolates from patients with CDI were analyzed, from July to August 2016, taken from hospitals in the Horne Povazie region of northern Slovakia. The aim of the study was also to identify hypervirulent strains (e.g., the presence of RT027 or RT176). METHODS: The retrospective analysis of biological samples suspected of CDI were analyzed by GDH, anaerobic culture, enzyme immunoassay on toxins A/B, multiplex "real-time" PCR and PCR capillary-based electrophoresis ribotyping, and by MALDI TOF MS. RESULTS: C. difficile isolates (n = 44) were identified by PCR ribotyping, which revealed five different ribotypes (RT001, 011, 017, 081, 176). The presence of hypervirulent RT027 was not identified. The C. difficile isolates (RT001, 011, 081, 176) were susceptible to metronidazole and vancomycin. One isolate RT017 had reduced susceptibility to vancomycin. A statistically significant difference between the most prevalent PCR ribotypes, RT001 and RT176, regarding variables such as albumin, CRP, creatinine, the length of hospitalization (p = 0.175), and glomerular filtration (p = 0.05) was not found. CONCLUSION: The results of PCR capillary-based electrophoresis ribotyping in the studied samples showed a high prevalence of RT176 and 001.

Cytotoxic effect of aminoglycoside antibiotics on the mammalian cell lines.

Aminoglycoside antibiotics have been used for treating serious but also routine infections in veterinary and human medicine for many years. The basic aim of this work is to evaluate the cytotoxicity of dihydrostreptomycin and neomycin in vitro on three cell cultures - BHK-21 (Syrian golden hamster kidney fibroblast), VERO (African green monkey kidney fibroblast) and FEA (feline embryonic fibroblast) cells. The morphological changes were examined by Giemsa staining. Cells were dried and visualized under fluorescence microscope. After the exposure to different experimental doses of dihydrostreptomycin (812.5-20000 µg/mL) and neomycin (1000-20000 µg/mL) during 24 h, the viability of BHK-21, FEA and VERO cell lines were evaluated by MTT assay. Viability of BHK-21 cells significantly (P < 0.001) decreased after treatment with 3500; 5500 and 7500 µg/mL of dihydrostreptomycin and 9000; 10000 and 20000 µg/mL of neomycin. The FEA cell viability decreased significantly (P < 0.001; P < 0.01) at 2500 and 3000 µg/mL dihydrostreptomycin and at 3000 µg/mL of neomycin treatment. Only the highest concentration of dihydrostreptomycin (20000 µg/mL) reduced VERO cell viability significantly (P < 0.01). Based on or results we can assume the effect of different antibiotics in different concentrations on cell lines is various. Detection of antibiotic toxicity to animal cells is very important because of the increasing resistance of bacteria. One of the solutions is drug dose increasing, but only to a certain concentration, since the toxic effect over the therapeutic one will prevail, which we have also shown in this work.

Escherichia coli as a carrier of tetracyclines and penicillins resistance in wild pheasant (Phasianus colchicus).

Wild animals like pheasant seem to be a good source of information about human activities. Therefore, the wild pheasants and relative stable appendix microcenosis were selected for antibiotic resistance testing. Penicillin resistance by MALDI-TOF Mass Spectrometry and tetracyclines resistance by genetic methods using specific primers were tested. Differences between tetracycline and penicillin resistance were detected. Results showed high prevalence of resistant Escherichia coli isolated from wild pheasant appendix. E. coli isolated from wild pheasant appendix carried plasmids for penicillins and tetracyclines resistance where they were responsible for enzymatic degradation of penicillin and carried genes for regulating efflux pumps for tetracyclines. Results showed that tetracyclines and penicillins resistance is widespread between wild pheasants with a carrier as Escherichia coli isolated from relative stable microcenosis of appendix.

Antibiotics Versus Natural Biomolecules: The Case of In Vitro Induced Bacteriospermia by Enterococcus Faecalis in Rabbit Semen.

Male subfertility is a global issue in human reproduction as well as in animal reproduction. Bacterial infection and semen contamination are still widely overlooked. As the collection of ejaculates is not a sterile process, it is necessary to add antimicrobial agents to avoid a possible depreciation of semen samples. As traditionally used antibiotics have been questioned because of an ever-increasing bacterial resistance, natural bioactive molecules could offer an alternative because of their antibacterial and antioxidant properties. As such, we decided to compare the effects of selected natural biomolecules (resveratrol-RES, quercetin-QUE and curcumin-CUR) with routinely used antibiotics in animal biotechnologies (penicillin-PEN, gentamicin-GEN and kanamycin-KAN) on the rabbit sperm vitality in the presence of Enterococcus faecalis. Changes in the sperm structural integrity and functional activity were monitored at 0, 2, 4 and 6 h. Computer-assisted sperm analysis (CASA) was used for the assessment of spermatozoa motility. Production of reactive oxygen species (ROS) was evaluated using chemiluminiscence, while the mitochondrial membrane potential (ΔΨm) was examined using the JC-1 dye. Finally, the sperm chromatin dispersion (SCD) test was used to assess DNA fragmentation, and changes to the membrane integrity were evaluated with the help of annexin V/propidium iodide. The motility assessment revealed a significant sperm motility preservation following treatment with GEN (p < 0.001), followed by PEN and CUR (p < 0.01). QUE was the most capable substance to scavenge excessive ROS (p < 0.001) and to maintain ΔΨm (p < 0.01). The SCD assay revealed that the presence of bacteria and antibiotics significantly (p < 0.05) increased the DNA fragmentation. On the other hand, all bioactive compounds readily preserved the DNA integrity (p < 0.05). In contrast to the antibiotics, the natural biomolecules significantly maintained the sperm membrane integrity (p < 0.05). The microbiological analysis showed that GEN (p < 0.001), KAN (p < 0.001), PEN (p < 0.01) and CUR (p < 0.01) exhibited the strongest antibacterial activity against E. faecalis. In conclusion, all selected biomolecules provided protection to rabbit spermatozoa against deleterious changes to their structure and function as a result of Enterococcus faecalis contamination. Therefore, administration of RES, QUE and/or CUR to rabbit semen extenders in combination with a carefully selected antibacterial substance may be desirable.

Trace Metals in the Freshwater Fish Cyprinus carpio: Effect to Serum Biochemistry and Oxidative Status Markers.

Interactions between trace metals, serum biochemical parameters, and oxidative status markers were observed. Freshwater fish Cyprinuscarpio blood samples (n = 38) were collected at the beginning of May (n = 19) and at the end of July (n = 19) of 2015. The concentrations of metals (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Se, Sr, and Zn) were analyzed in blood serum samples of fishes by inductively coupled plasma optical emission spectrometry (ICP-OES), and Hg was determined by cold-vapor atomic absorption spectroscopy (CV-AAS). The general scheme of descending concentrations of metals in blood serum samples was as follows: Zn > Fe > Cu > Sr > Cr > Ni > Mn > Pb > Se > As > Cd > Hg. Zn was the most accumulated element (4.42-119.64 mg/L) in both seasons. Overall, the trace element content was higher in spring season, except Hg, Ni, Se, and Sr. The seasonal effect was confirmed for Mn, Zn, Mg, Glu, AST, and Chol levels and for most oxidative status markers. The gender effect was confirmed for Sr, GPx, PC, Chol, and CK concentrations. Trace metals (especially Cd, Cr, Cu, Fe, Hg, Mn, Ni, Sr, Zn, As) significantly affected some blood serum chemistry parameters. The correlation analysis between oxidative status markers (ROS, TAC, MDA, SOD, GSH, UA, BHB, and Alb) and trace metal (Cd, Cu, Ni, Sr, Hg, Pb, Fe, Mn) content confirmed statistically significant interactions in both seasons. Obtained results indicate specific actions of trace metals.

Antimicrobial activity of actinomycetes and characterization of actinomycin-producing strain KRG-1 isolated from Karoo, South Africa

In the present study we reported the antimicrobial activity of actinomycetes isolated from aridic soil sample collected in Karoo, South Africa. Eighty-six actinomycete strains were isolated and purified, out of them thirty-four morphologically different strains were tested for antimicrobial activity. Among 35 isolates, 10 (28.57%) showed both antibacterial and antifungal activity. The ethyl acetate extract of strain KRG-1 showed the strongest antimicrobial activity and therefore was selected for further investigation. The almost complete nucleotide sequence of the 16S rRNA gene as well as distinctive matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) profile of whole-cell proteins acquired for strain KRG-1 led to the identification of Streptomyces antibioticus KRG-1 (GenBank accession number: KX827270). The ethyl acetate extract of KRG-1 was fractionated by HPLC method against the most suppressed bacterium Staphylococcus aureus (Newman). LC//MS analysis led to the identification of the active peak that exhibited UV-VIS maxima at 442 nm and the ESI-HRMS spectrum showing the prominent ion clusters for [M-H2O+H]+ at m/z 635.3109 and for [M+Na]+ at m/z 1269.6148. This information could be assigned to chromopeptide lactone antibiotic - actinomycin. Our results suggest that unexplored soils could be an interesting source for exploring antibacterial secondary metabolites.

Trace elements content in semen and their interactions with sperm quality and RedOx status in freshwater fish Cyprinus carpio: A correlation study.

Objective of the present study was to investigate interactions between trace elements content and RedOx status, as well as sperm quality parameters (motility features, DNA fragmentation) in fish spermatozoa in natural conditions. Reproductively mature male freshwater fish (n = 16) of Cyprinus carpio breed were used in the study. Trace elements content was determined in fish milt samples by inductively-coupled plasma optical emission spectrometry (ICP-OES) and by cold-vapor atomic absorption spectroscopy (CV-AAS). Sperm quality evaluation was realized by computer-assisted sperm analysis (CASA) quantifying several parameters: concentration, total motility, progressive motility, distance average path, distance curved line, distance straight line, velocity average path, velocity curved line, velocity straight line, straightness, linearity, amplitude of lateral head displacement and beat cross frequency. The general scheme of descending concentrations of trace metals in semen samples was following: Zn > Fe > Cu > As > Sr > Ni > Mn > Se > Pb > Cr > Cd > Hg. Total motility of spermatozoa was relatively high (91.45%), however progressive motility was not even half of this value (39.47%). Sperm DNA fragmentation values were relatively low (4.00-6.29%). The percentage of immotile spermatozoa showed a significant correlation with all RedOx status parameters and also with DNA fragmentation. Positive statistically significant correlations were observed between trace elements (Mn, Se, Sr, and Zn) and some qualitative spermatozoa parameters (velocity and distance parameters). Cu and Hg content shows similar negative associations with progressive motility. Hg also interacted with production of malondialdehyde. Overall, the present study suggests application of multi-component mixtures of environmentally related trace elements concentrations when assessing the potential reproductive risk.

Rapid identification of Streptomyces tetracycline producers by MALDI-TOF mass spectrometry.

The main objective of this study was using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for assembling of DSM (German Collection of Microorganisms) Streptomyces spectral database and identification of wild Streptomyces cultures, which were clustered by MALDI-TOF Biotyper OC software as well as for teracycline detection by observing of obtained spectra using flexAnalysis software. Production of tetracycline was confirmed by thin-layer chromatography. Presence of tetracycline mass spectrum was verified by several tetracycline producers (Streptomyces aureofaciens LMG 5968, S. aureofaciens 84/25, and S. aureofaciens BMK) and by pure tetracycline mass. Our results showed that it is possible to use MALDI-TOF MS for identification of tetracycline producers within Streptomyces genera by several easy steps. The purpose of this study was to establish cheap and quick detection of tetracycline producers.

Streptomyces globosus DK15 and Streptomyces ederensis ST13 as new producers of factumycin and tetrangomycin antibiotics.

Fifty seven soil-borne actinomycete strains were assessed for the antibiotic production. Two of the most active isolates, designed as Streptomyces ST-13 and DK-15 exhibited a broad range of antimicrobial activity and therefore they were selected for HPLC fractionation against the most suppressed bacteria Staphylococcus aureus (ST-13) and Chromobacterium violaceum (DK-15). LC/MS analysis of extracts showed the presence of polyketides factumycin (DK15) and tetrangomycin (ST13). The taxonomic position of the antibiotic-producing actinomycetes was determined using a polyphasic approach. Phenotypic characterization and 16S rRNA gene sequence analysis of the isolates matched those described for members of the genus Streptomyces. DK-15 strain exhibited the highest 16S rRNA gene sequence similarity to Streptomyces globosus DSM-40815 (T) and Streptomyces toxytricini DSM-40178 (T) and ST-13 strain to Streptomyces ederensis DSM-40741 (T) and Streptomyces phaeochromogenes DSM-40073 (T). For the proper identification, MALDI-TOF/MS profile of whole-cell proteins led to the identification of S. globosus DK-15 (accession number: KX527570) and S. ederensis ST13 (accession number: KX527568). To our knowledge, there is no report about the production of these antibiotics by S.globosus and S. ederensis, thus isolates DK15 and ST13 identified as S. globosus DK-15 and S.ederensis ST-13 can be considered as new sources of these unique antibacterial metabolites.

Resistance in bacteria and indirect beta-lactamase detection in E. coli isolated from Culex pipiens detected by matrix-assisted laser desorption ionization time of flight mass spectrometry.

The aim of this study was detections of antibiotic resistance and resistance mechanism in bacteria isolated from mosquitos (Culex pipiens) living near humans. Therefore, antibiotic resistance in bacteria isolated from Culex pipiens was investigated by disk diffusion test and MIC E-test in this study. MALDI-TOF mass spectrometry was used for detection of resistant mechanism. In this study, hydrolytic breakdown products after a few hours of incubation of the bacteria isolated from Culex pipiens were detected. Results show that enzymatic destruction of ampicillin by beta-lactamases is able to be detected by MALDI-TOF mass spectrometry from wild strains of potential pathogens. The MALDI-TOF mass spectrometry is useful method for routine detection of beta-lactamases resistant mechanism, but overnight incubation of pure culture is necessary. The results are important for proper and fast intervention to limit the spread of beta-lactamase-producing wild bacteria and provide information for appropriate initial therapy of the infections caused by these microbes.