Identification of the contaminating psychrotrophic bacteria in refrigerated bulked raw milk and the assessment of their deteriorating potential

This work aimed to characterize, identify, and determine the deteriorating potential of the contaminating psychrotrophic bacteria in refrigerated raw milk. Samples were submitted to serial dilutions and plated in specific culture media to form a bacterial culture collection. The isolates were characterized for their morphology and biochemical characteristics. The deteriorating potential of the isolates was determined according to the proteolytic, lipolytic and lecithinase activities at 4.0 ºC, 6.5 ºC, and 25.0 ºC. The results obtained for deterioration potential were assessed by the multivariate statistical method and by the principal components analysis (PCA). A total of 159 isolates were characterized, and of these, 46 strongly proteolytic Gram-negative isolates were selected for identification using the API 20 NE kit. The predominant bacteria were Gram-negative and oxidase and catalase positive, with a predominance of bacteria of the genus Pseudomonas. Using PCA, it was shown that the bacteria with the greatest deterioration potential were lecithinase producers, and that, in the autumn, proteolytic bacteria predominated at 4.0 ºC. Of the 46 isolates identified, more than 80% belonged to the species Pseudomonas fluorescens. Thus, attention should be given to the importance of implementing microbial contamination prevention measures in the bulking process, since, even under refrigeration, psychrotrophic bacteria multiply and produce enzymes that deteriorate lipids and proteins, with consequent quality losses of the milk and its derivatives, yield losses in the production of dairy products, and economic losses.

Nodulation Compatibility and Symbiotic Performance of Rhizobia spp. With Different Landraces of Bambara Groundnut (Vigna Subterranea (L.) Verdc.) Collections

The symbiosis of the legume bambara groundnut (Vigna subterranean L.Verde) with its rhizobial partners has not been studied sufficiently compared to several other legumes throughout Africa. In this study, a nodulation compatibility screening was conducted on 16 different landraces of this legume using five Rhizobia strains previously isolated from active nodules of Desmodium uncinatum, Arachis hypogaea, Cyamopsis tetragonoloba, Glycine max and Phaseolus vulgaris and deposited at the South African Rhizobium Culture Collection (SARCC). A screening assay was conducted under glasshouse to select compatible rhizobia strains that nodulate and enhance growth in one or more genotypes of V. subterranean (L.) Verdc. Pre-germinated seeds of each landraces planted in sterile river sand medium were inoculated with 108 cfu ml-1 of the rhizobial strains (2ml/seed) and monitored with regular watering for six weeks. Parameters such as nodule number, nodule color and positions, plant biomass were determined in test genotypes. Significant differences were observed among landraces in nodule number and plant biomass, and among rhizobial strains in nodule number. Principal component analysis (PCA) showed that root nodule rhizobia strains SARCC-388 and SARCC-578 characterized as Bradyrhizobium zhangiangens and Bradyrhizobium centrosematis, respectively exhibited the highest nodulation compatibility with one or more bambara groundnut landraces. This study demonstrated that many of the bambara landraces did not show nodulation preference to a unique group of rhizobia, confirming that V. subterranean (L) Verdc can be nodulated by more than one species of rhizobia, especially by rhizobia belonging to the cowpea miscellany cross inoculation group.

Bradyrhizobium japonicum FN1 produces an inhibitory substance that affects competition for nodule occupancy

Bacteriocins are narrow spectrum antibiotics of bacterial origin that can affect competition in resource-limited environments such as the rhizosphere. Therefore, bacteriocins may be good candidates for manipulation in order to generate more competitive inocula for soybean. In this study,<i> B. japonicum</i> FN1 along with other Bradyrhizobia in our culture collection were screened for bacteriocin-like activity. A total of five distinct inhibitory activities could be observed. FN1 genes putatively involved in bacteriocin production were computationally identified. These genes were mutagenized and the subsequent strains were screened for loss of inhibitory activity. Mutant strain BRJ-48, with an insert in<i> bjfn1_01204</i>, displayed a loss of the ability to inhibit an indicator strain. This loss could be complemented by the introduction of a plasmid expressing <i>bjfn1_01204 </i>in trans. The strain carrying the mutation did not affect competition in broth cultures, but was shown to be less competitive for nodule occupancy. Annotation suggests that <i>bjfn1_01204</i> encodes a carboxymuconolactone decarboxylase, however the direct contribution of how this enzyme contributes to inhibiting the tester strain remains unknown.

A Retrospective Characterization of Dexmedetomidine-Suspected Fever and Its Consequences in Adult Critically Ill Patients

Background: Current evidence for dexmedetomidine-suspected fever (DSF) is limited. Lack of recognition may lead to costly or potentially harmful interventions for critically ill patients. Objective: The primary objective was to characterize escalations of care related to DSF. Secondary objectives were to describe the incidence, severity, and consequences associated with DSF. Methods: A retrospective review was conducted in critically ill adults who developed fever ≥39°C within 12 h from initiation of dexmedetomidine, with resolution of fever to <39°C within 12 h after discontinuation. The primary outcome was percentage of patients who received an escalation of care due to fever. Secondary outcomes included the percentage of patients who developed a multidrug-resistant organism or Clostridium difficile infection. Results: Eighteen of 3943 patients screened in 4099 encounters met criteria for DSF (0.4%). The majority were white (83.3%), male (66.7%), and underwent cardiac surgery (61.1%). Median (interquartile range [IQR]) time to fever onset and resolution were 5.5 (3.6-7.6) and 1.3 (1.0-2.9) h. Nine patients (50%) underwent infectious workup including antimicrobial initiation (n = 1, 5.6%), broadening of antimicrobials (n = 4, 22.2%), or culture collection (n = 9, 50%). Eleven patients (61.1%) underwent attempted temperature reduction. Twelve patients (66.7%) underwent diagnostic imaging. Incidence of multidrug-resistant organism and C. difficile infection were low (11.1 and 16.7% of fever patients, respectively). Conclusion and Relevance: Incidence of DSF was low and more common in cardiac surgery patients. Unrecognized DSF led to an escalation of care in most patients. Dexmedetomidine exposure should be considered as a potential cause of fever in critically ill adults.

Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses.

Characterization of Morganella sp. for its Paraquat Degradation Potential

One of the beneficial roles of the microbial population is their ability to convert toxic herbicides to lesser toxic compounds such as water and carbon (IV) oxide. Paraquat which is an acutely toxic herbicide is used on farmlands and has been found to affect human health. This study was aimed at characterizing bacteria with the potential to degrade paraquat. Previously isolated bacteria from culture collection labelled A-F were screened for their potential to degrade and utilized paraquat as the sole carbon source in Bushnell Hass agar media. Of the six isolates, isolate E (Morganella sp.) was observed to have the highest growth and tolerance to paraquat after 72 h of incubation at 37 ºC. Characterization study revealed that Morganella sp. can utilize and grow with optimum conditions of pH 6.5, the temperature of 30 ºC and can tolerate up to 400 mg/L paraquat concentration with an increase in growth as inoculum size increases. Thus, these findings showed that Morganella sp. can degrade toxic paraquat to a less toxic form and therefore can be a good isolate for the future bioremediation process of the pollutant.

Characterization of Butachlor Degradation by A Molybdenum-Reducing and Aniline-degrading Pseudomonas sp.

Butachlor is a chloroacetanilide herbicide that is selective in action and commonly used for pre-emergence control of weeds. It is believed to have range of toxicity from acute to chronic and also can cause DNA strand breaks and chromosomal aberrations in humans. This study was aimed at characterizing the potential of previously isolated bacteria for butachlor degradation. The isolates from culture collection, labelled A-F were screened for butachlor degradation on Bushnell Hass agar media with butachlor as a sole carbon source. Isolate A (molybdenum-reducing and aniline-degrading Pseudomonas sp.) was observed to grow best and tolerated the highest concentration of butachlor supplemented in the media after 72 h of incubation at 37 ℃. Characterization study revealed that the Pseudomonas sp. can utilize and grow with butachlor at optimum pH between 6.0 - 6.5, temperature between 30 – 37 ℃ and can tolerate up to 600 mg/L butachlor concentration with increase in growth as inoculum size increases. Additionally, this bacterial strain shows no lag phase regardless of the concentration of the herbicide used and reach its maximum growth after 24 h of incubation. The ability of this isolate to tolerate and utilize butachlor as sole carbon source makes it suitable for future bioremediation of this toxicant.

The antioxidant activity of extracts of the mycelium and the culture fluid of medicinal macromycetes of Pholiota (Fr.) P. Kumm. genus

Aim. To determine the value of the antioxidant activity of the biomass and culture fluid extracts of strains of seven species of Pholiota genus: P. adiposa, P. alnicola, P. aurivella, P. limonella, P. nameko, P. squarrosa, P. subochracea, which stored in the Mushroom Culture Collection (IBK) of the M. G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine.Materials and methods. The antioxidant properties of the biomass and culture fluid extracts of strains of Pholiota genus were determined by the method of Elfahri et al. using DPPH (1,1-diphenyl-2-picrylhydrazyl). Mycelia of the strains studied were grown by the surface method on a liquid glucose-pepton-yeast medium. The culture fluid was separated from the mycelial biomass by filtration through a capron filter. The absorption of methanol extracts of the culture fluid and the biomass of the strains studied was measured at 517 nm on a SF 46 LOMO spectrophotometer.Results and discussion. Comparing the data obtained we can conclude that the antioxidant effect is significantly higher in the case of methanol biomass extracts – the indicators ranged from 65.98 ± 0.98 % (P. nameko) to 83.6 ± 1.4 % (P. alnicola). As for the culture fluid extracts, the maximum values were recorded in the case of P. limonella (38.3 ± 1.14 %), and the minimum values were observed for P. subochracea (7.37 ± 0.46 %).Conclusions. For the first time, the value and limits of variation in the antioxidant activity of the biomass (65-83 %) and culture fluid extracts (7.4-38 %) have been determined for strains of medicinal fungal species P. adiposa, P. alnicola, P. aurivella, P. limonella, P. nameko, P. squarrosa, P. subochracea.

First Report of Anthracnose Caused by Colletotrichum acutatum On Olive Fruits in Pakistan

Anthracnose symptoms on olive (Olea europaea) fruits cv. “Gamlick” were found in farmer orchards in Chakwal, Punjab, Pakistan (32° N and 72° E), with an average prevalence of 59%. Fruit symptoms start as thin, black, sunken lesions with a watery appearance that grow in diameter and coalesce into a large sunken soft zone. Lesions on mature fruit become noticeable in 5 to 6 days after infection, if temperatures are favorable (28°C). On the fruit lesion, orange conidial masses in dispersed or concentric circle arrangement can appear. Fragments (5 mm) were taken from the margins of fruit lesions and surface-sterilized with 70% ethanol (1 min) and 1% NaClO (2 min), cleaned with sterile purified water, blotted dry, and plated on potato dextrose agar (PDA) in Petri dishes. The petri plates were incubated at 27°C. A fungus was consistently isolated, and thirty-five isolates were characterized. Aerial mycelia from olive isolates Colonies were compact, initially white or cream white, then grey, and eventually dark grey, with conidium masses forming in the middle. Mycelium is branched, septate, and hyaline. Conidia are hyaline, aseptate, fusiform, or often cylindrical, with obtuse apices and tapering bases. Their mean size was 8.5µm in length and 3.0 µm in width. Based on morphological features, the fungus was tentatively identified as Colletotrichum acutatum (Agosteo G.E., 2010). The identification was confirmed by amplification and sequencing of a representative isolate's internal transcribed region (ITS), Beta- tubulin region (TUB2), Actin region (ACT), and Glyceraldehyde 3-phosphate dehydrogenase region (GAPDH) with the primers ITS1/ITS4 (Gardes & Bruns 1993), TUB4/TUB5 (Woudenberg et al. 2009), ACT1/ACT3 (Carbone & Kohn 1999) and GDF1/GDR1 (Guerber et al. 2003). BLAST analysis revealed 100% identity for ITS, GAPDH and ACT and 99% identity for TUB, between the sequences of the olive fruit isolate (GenBank Accessions MW647502, MZ436968, MZ714412 and MW810331, respectively) and sequences of C. acutatum reference isolates (GenBank Accessions GO613492, KF975660.1, MT274752.1 and MH547616 respectively). Phylogenetic analysis based on ITS, GAPDH and TUB regions of the olive fruit isolates and reference isolates of various Colletotrichum species using the MEGA X software program confirmed the isolate from olive was C. acutatum. The fungal isolate was deposited as a living culture in the Barani Agricultural Research Institute's fungal culture collection center (BACA.9381). Pathogenicity tests were conducted with this isolate by placing a 20 µl drop of a conidial suspension (3 × 107conidia ml−1) on five healthy olive cv. Gemlik fruits. As a control, five non-inoculated olive fruits were used. Fruits were placed at a temperature of 27°C with artificial light and a photoperiod of 12 hours. Anthracnose symptoms developed only on inoculated fruits after seven days of inoculation. The fungus was re-isolated from symptomatic fruits, and its identity was confirmed through morphological characteristics, thus verifying Koch's postulates. To the best of our knowledge, this is the first report of C. acutatum infecting olive fruits in Chakwal region of Pakistan.

A Round Trip to the Desert: In situ Nanopore Sequencing Informs Targeted Bioprospecting

Bioprospecting expeditions are often performed in remote locations, in order to access previously unexplored samples. Nevertheless, the actual potential of those samples is only assessed once scientists are back in the laboratory, where a time-consuming screening must take place. This work evaluates the suitability of using Nanopore sequencing during a journey to the Tabernas Desert (Spain) for forecasting the potential of specific samples in terms of bacterial diversity and prevalence of radiation- and desiccation-resistant taxa, which were the target of the bioprospecting activities. Samples collected during the first day were analyzed through 16S rRNA gene sequencing using a mobile laboratory. Results enabled the identification of locations showing the greatest and the least potential, and a second, informed sampling was performed focusing on those sites. After finishing the expedition, a culture collection of 166 strains belonging to 50 different genera was established. Overall, Nanopore and culturing data correlated well, since samples holding a greater potential at the microbiome level also yielded a more interesting set of microbial isolates, whereas samples showing less biodiversity resulted in a reduced (and redundant) set of culturable bacteria. Thus, we anticipate that portable sequencers hold potential as key, easy-to-use tools for in situ-informed bioprospecting strategies.