Accurate enumeration of Salmonella enterica and Shiga-toxigenic Escherichia coli (STEC) is critical for validating pathogen control in dry fermented sausage production. Lactose-Sulfite-Phenol Red-Rifampicin (LSPR) agar is a selective medium designed to recover and enumerate these pathogens under environmental stress. S. enterica produces black colonies on LSPR, while STEC produces cream-colored colonies, enabling visual differentiation. This study evaluates LSPR’s performance for recovering S. enterica and STEC after lactic acid (pH 3.0) and salt (35.9 g NaCl/100 mL) stress and its suitability for co-enumeration in sausage batter.

Cocktails of seven rifampicin-resistant serotypes of S. enterica and STEC were stressed for 0, 30, 60, and 120 seconds, then plated on BHI, LSPR, and modified LSPR (MLSPR). Initial bacterial loads averaged 7.5 log CFU/mL. After 120 seconds of acid stress, STEC declined by 4.2 log CFU/mL, while S. enterica declined by 3.1 log CFU/mL (p < 0.05). Under salt stress, reductions were 2.8 and 2.1 log CFU/mL, respectively (p > 0.05). Two-way ANOVA revealed that exposure time significantly affected bacterial survival (p = 0.0027), with no significant differences in recovery between media types (p = 0.0776). MLSPR did not enhance recovery over LSPR (p = 0.238).

The interaction between media and exposure time was not statistically significant for either acid (p = 0.2380) or salt stress (p = 0.9031), indicating consistent bacterial reductions across media.

The findings indicate that media type had no statistically significant effect on bacterial counts, as LSPR, BHI, and MLSPR supported similar levels of pathogen recovery showing the suitability of the media for co-enumeration of Salmonella and STEC in sausage batter. However, exposure time to stress solution showed a significant impact on bacterial survival, with longer exposure times resulting in greater reductions in STEC and S. enterica counts.

Introduction: Visceral Leishmaniasis represents a significant public health challenge in Brazil, characterized by its zoonotic nature and potential for severe clinical outcomes. The distribution and dynamics of this disease can be influenced by various factors, including environmental, social, and economic changes. Understanding its epidemiological trends is essential for guiding effective prevention and control efforts. Methods: A cross-sectional, descriptive, retrospective, and quantitative study was conducted on confirmed cases of Visceral Leishmaniasis in the five regions of Brazil during the pre-pandemic period (2016 to 2019) and the pandemic period (2020 to 2023). The data were obtained from the Department of Informatics of the Unified Health System (DATASUS), specifically from the "Notifiable Diseases and Conditions" section. Results: During the pre-pandemic period (2016–2019), a total of 14,589 cases of Visceral Leishmaniasis were reported in Brazil, with a 46% overall reduction during the pandemic period (2020–2023). The decrease in the number of cases varied across the country's regions, with the North region showing the largest decline of 56%. The Northeast experienced a 44% reduction, followed by the Southeast with a 52% decrease. In the South, the reduction was 16.9%, and in the Central West, it was 6.1%. Conclusions: Based on data analysis, it is possible to infer that the COVID-19 pandemic had an impact on the reporting of Visceral Leishmaniasis cases in Brazil. The sharp decline in notifications during the pandemic, particularly in the Northern region, suggests the possibility of an overburdened healthcare system, reduced demand for diagnosis and treatment due to social isolation, and a decrease in control programs and epidemiological surveillance. Therefore, it can be proposed that the reduction in Visceral Leishmaniasis cases is associated with lower case detection and reporting.

Ilex paraguariensis (Ip) is a crop with economic and cultural importance in South America, where its leaves are used to prepare an infusion called “mate”. This crop is affected by different phytopathogenic microorganisms. Cylindrocladium (Cy) are the causal agent of the black spot of Ip. The objective of this work was to evaluate the use of two thyme (Thymus vulgaris) essential oils (EOs) as control agents of Cy. The thyme EOs (T1 and T2) were characterized by means of GC-MS. Cy was isolated from infected leaves of Ip. Cy, and a 14-day culture was used to obtain the work solution (100,000 spores/ml). The EOs were diluted in dimethyl sulfóxide at a rate of 1:4, and nine two-fold serial dilutions were performed. Treatments consisted of the addition of 15 µl of work solution to 165 µl broth with Ip and 20 µl of each EO dilution, which were then incubated for 72 h. Four replications were carried out. Negative (Ip broth) and positive controls (without EOs) were performed. The minimum inhibitory concentration (MIC) was determined as the minimal concentration in which no micelial growth was observed. Tubes whitout micelial growth were sowed in Ip medium and incubated for 48 h. The minimum fungicide concentration (MFC) was defined as the minimal EO concentration at which no Cy colonies were grown. T1 was mainly composed of thymol (40.32%) and p-cymene (36.43%), while T2 comprised p-cymene (59.65%), thymol (11.78%), and eucalyptol (5.35%). Both EOs exhibited fungistatic and fungicidal activity, with MIC values of 2.35 µl/ml for T1 and 31.25 µl/ml for T2, and MFCs of 2.93 µl/ml and 50 µl/ml, respectively. Despite both EOs showing good antifungic activity, T1 showed a greater bioactivity, which was correlated with the higher relative amount of thymol compared with T2. These results demonstrate the effectiveness of thyme EOs as natural control agents against Cy.

Overgrazing and climate change have resulted in the expansion of marginal lands and the deterioration of soil quality, leading to a decline and extinction of numerous Fabaceae species. In this context, particular attention was given to study the symbiotic interactions between native legumes and their associated microbiome, as a sustainable alternative for restoring vegetation cover and reestablishing soil fertility in vulnerable areas. The main objective of this study was to isolate and characterize the root nodules endophytes from 15 wild legumes, originating from diverse Tunisian climatic zones (humid, arid, and Saharan). The taxonomic diversity of the isolates was determined by means of morphological, genomic, and phylogenetic analyses based on molecular screening by BOX-PCR and sequencing of 16S rRNA and atpD genes. To further assess the symbiotic characteristics of certain isolates, we proceeded with the amplification of nodC and nodZ genes. In a collection of 210 strains, phylogenetic analysis revealed the presence of 46 rhizobial strains distributed among 6 genera, namely Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium, Neorhizobium, and Agrobacterium. Our results showed the predominance of Sinorhizobium genus in arid and Saharian zones of southern Tunisia, represented by three different symbiovars: sv. meliloti, sv. Tunisiaense, and sv. Vachelliae. However, no nodC genes were detected in Bradyrhizobium, which was only present under humid climate suggesting the impact of environmental conditions on the diversity of the rhizomicrobiome and its symbiotic interaction with wild legumes. In addition, various non-rhizobial isolates were identified, including strains from α-, β-, and Gamma-proteobacteria, Firmicutes, and Actinobacteria. These strains were not only highly tolerant to different abiotic stresses, but also presented different plant growth promoting features (PGP), making them perfect candidates to boost the survival and productivity of food legumes in drylands.

Introduction: Pseudomonas aeruginosa (P. aeruginosa) is a multidrug-resistant opportunistic pathogen frequently encountered in clinical settings, posing substantial challenges for effective treatment. Its resistance often arises from mutations in critical drug targets, such as penicillin-binding protein-3 (PBP3), a key β-lactam target essential for bacterial survival. The F533L mutation is linked to resistance against multiple antibiotics, underscoring the need for alternative therapeutic strategies. Methods: This study employed molecular docking to analyze the interactions between zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs) and the mutated PBP3 (F533L) of P. aeruginosa. Additionally, computational approaches were used to assess the impact of the F533L mutation on the structural stability of PBP3. Binding energy and interaction analyses were conducted to evaluate the PBP3-nanoparticle complexes. Results: The F533L mutation was found to alter the stability of PBP3, potentially contributing to resistance mechanisms. Molecular docking revealed that ZnONPs and CuONPs demonstrated binding energies of -2.15 kcal/mol and -1.48 kcal/mol, respectively. Both nanoparticles formed hydrogen bonding interactions with the mutated residue 533L, highlighting their potential to mitigate resistance. Conclusions: The findings suggest that ZnO and CuO nanoparticles could serve as promising therapeutic agents against antibiotic resistance in P. aeruginosa, particularly targeting the F533L mutation in PBP3. Further experimental and in vitro studies are essential to validate their therapeutic efficacy and broader applications.

Endophytes reside in healthy plant tissue without causing visible harm. Fungal endophytes produce structurally and chemically diverse secondary metabolites, including those similar to the host plant, with antimicrobial and anti-inflammatory activities. Fungal endophytes also produce various extracellular enzymes with applications in various industries including the pharmaceutical industry. Despite this knowledge, the diversity and bioactivities of fungal endophytes of South African medicinal plants are relatively underexplored. Thus, the aim of this study was to isolate fungal endophytes from the medicinal plant Monsonia angustifolia and evaluate their antimicrobial and extracellular enzymatic activities. A total of 29 morphologically distinct fungal endophytes with 21 isolates related to filamentous fungi and 8 to yeasts were isolated from the surface-sterilised leaves, stems and spikes. The majority of the isolates (59%) were obtained from the stems. The sequencing of the internal transcribed spacer region revealed that all the filamentous fungi and four yeast isolates belonged to the Phylum Ascomycota, while the other four yeast isolates reside with Basidiomycota. The broth micro-dilution assay revealed that the ethyl acetate crude extracts of the endophytes possessed varying inhibitory activity, with minimum inhibitory concentrations ranging from 0.02 to 2.50 mg/mL against six clinically significant pathogens. Agar-plate screening for amylase, cellulase, laccase, lipase, pectinase, protease and xylanase enzymes revealed that the isolates had the ability to produce at least one of the tested enzymes, with four filamentous fungi exhibiting the ability to produce all the screened enzymes. This study highlights the significance of fungal endophytes as sources of antimicrobial secondary metabolites and industrially significant enzymes.

Introduction

The need for alternative techniques of microbial control has been determined by the rapid spread of antibiotic-resistant bacteria. One approach that is gaining popularity is antimicrobial photodynamic therapy (aPDT). This method involves the use of a chemical compound (photosensitizer), which is activated by light in the presence of molecular oxygen. The activated photosensitizer induces the production of reactive oxygen species (ROS), which leads to cell destruction. Significant attention has been directed toward the application of 5-aminolevulinic acid (5-ALA) in aPDT. This compound occurs naturally in cells, where it is converted mainly to the intracellular photosensitizer protoporphyrin IX (PpIX). Over time, PpIX is transformed into heme (which does not possess photosensitizing properties) by the ferrochelatase enzyme [1]. An increased concentration of PpIX in cells can be achieved by inhibiting ferrochelatase with divalent metal ions (e.g, Mn^{2 +,} Zn^{2 +}) [2].

Methods

The aim of this study is to evaluate the combined effect of 5-ALA (2.5 mM), light, and metal ions on the photosensitization of bacteria. The microorganism tested was antibiotic-resistant Proteus mirabilis (PCM 543). Irradiation was performed with a 404 nm diode laser (the light dose was 23.5 J cm-2). For the research, ions of manganese (1 mM), zinc (1 mM), calcium (10 mM), and magnesium (10 mM) were used.

Results

The most effective approach was found to involve the use of 5-ALA with zinc ions, resulting in the destruction of almost 99% of bacterial cells, while the use of 5-ALA alone led to the eradication of approximately 90% of cells after the same exposure time.

Conclusions

The results obtained suggest that metal ions, especially zinc, may compete with iron ions for the ferrochelatase binding site, leading to the inhibition of the enzyme and, at the same time, the accumulation of PpIX in cells.

[1] doi:10.3390/ijms25073590

[2] doi:10.1074/jbc.M803372200

Bluetongue (BT) is an infectious disease of domestic and wild ruminants caused by Bluetongue virus (BTV), an arbovirus of the Orbivirus genus within Sedoreoviridae family. It’s primarily transmitted by biting midges of the Culicoides genus. BTV poses a significant threat to the livestock industry, particularly sheep. To date, a total of 36 serotypes of BTV have been characterized worldwide, causing periodic outbreaks, occurring most frequently in the Mediterranean basin. The control of BT disease is based on vaccination and several vaccines have been developed. Live attenuated vaccines and inactivated vaccines are widely used to prevent BT. However, despite their demonstrated efficacy, those vaccines have several limitations, including safety concerns and incomplete cross-protection among BTV serotypes. Recombinant subunit BT vaccines based on BTV structural proteins or virus-like particle vaccines (VLPs) may solve some of these limitations. In this study, we aim to design and develop vaccines that constitute an alternative to conventional attenuated vaccines. For this, we intend to express the VP2 and VP5 BTV antigens in Saccharomyces cerevisiae, a GRAS microorganism, by fusing the antigens to the secretion and cell wall retention signals of the Pir4 yeast cell wall protein. The VP2 and VP5 BTV structural proteins will be expressed in S. cerevisiae, targeting them to the yeast cell surface, or to the culture medium. The possibility of obtaining virus-like particles (VLPs), mimicking the virus, will also be studied.

Given that the research is currently underway, cloning, gene confirmation, sequencing and transformation of the constructs into the yeast expression system have been successfully completed. The confirmation of VP2, VP5, VLPs expression and assembly is currently under investigation, using immunofluorescence and Western blot analysis. The assessment of antigenicity and evaluation of the potential immunogenicity will be performed to validate the activity of the vaccine candidate in animals.

Antimicrobial resistance (AMR) poses a significant public health challenge, as AMR bacteria and their genes persist in environments such as farms and water systems. AMR bacteria can infect humans through contaminated food, water, or direct contact. We hypothesize that both intermittent exposure to clinical antimicrobials and environmental residues might exacerbate this colonization, leading to gut dysbiosis and facilitating the spread of resistance genes. This could enhance the survival and persistence of AMR bacteria and promote AMR gene transfer among bacterial communities in the gut.

To investigate this, we utilized a mouse model to examine the conjugative transfer of a mobile AMR plasmid from a Salmonella Heidelberg donor to Enterobacteriaceae in the gut under treatments of intermittent clinical doses of ampicillin and sub-clinical levels of tetracycline in drinking water. Fecal samples were collected from mice at regular intervals to isolate AMR bacteria on selective agar media. DNA was extracted from these samples for future 16S rRNA sequencing to analyze microbial community shifts and qPCR targeting ARGs to examine plasmid transfer dynamics.

Preliminary results show that the donor Salmonella Heidelberg persisted for four months in all treatment groups except the control, which had no antimicrobial exposure. The donor reached a high density immediately after each ampicillin treatment and gradually became undetectable. The AMR plasmid was transferred to commensal E. coli and to an introduced recipient Salmonella Heidelberg. While detailed sequencing and bioinformatics analyses are pending, initial findings highlight the significant impacts of antimicrobial exposure on the persistence of AMR bacteria and the transfer of AMR plasmids within the gut microbiota.

Covasna County in Romania is regarded as an agricultural region due to the exceptionally high proportion of arable land. One of the most significant indicators of agricultural productivity is soil fertility, which is influenced by numerous factors. Among these, the most important is the biological condition of the soil.

In our research, we analyzed the biological activity of soils in the surroundings of Sfântu Gheorghe. Soil samples were collected from five different soil types (preluvosol, faeoziom, cambic faeoziom, aluviosol and gleysol) in autumn 2018 and spring 2019. For each field, 20 samples were collected and homogenized, and the biological activity was measured in three replications using the fluorescein diacetate (FDA) hydrolysis method.

The faeoziom soil type showed an enzymatic activity of 3.9631±0.1818 µg/g*h in autumn and 2.9788±0.3277 µg/g*h in spring; the cambic faeoziom soil type showed an activity of 4.0072±0.1145 µg/g*h in autumn and 3.0548±0.1621µg/g*h in spring; the gleysol type showed an activity of 3.2869±0.1817 µg/g*h in autumn and 2.9988±0.4679 µg/g*h in spring; the aluviosol soil type showed an activity of 2.4186±0.0616 µg/g*h in autumn and 1.3421±0.3779 µg/g*h in spring; and the preluvosol type recorded the highest values, with 5.4517±0.2609 µg/g*h in autumn and 5.3677±0.1548 µg/g*h in spring. For the faeoziom, cambic faeoziom, and aluviosol soil types, an approximately one-unit decrease was observed in spring due to the lower temperatures during the spring sampling period. For the gleysol soil type, the decrease was much smaller, and the differences between the values were not statistically significant. We attribute this to the application of manure by farmers after the autumn sampling, as manure contains a considerable number of microorganisms. For the preluvosol soil type, enzymatic activity also decreased slightly, which can be explained by the fact that this area is a pastureland with continuous vegetation cover and no soil disturbance, providing a stable habitat for microorganisms, andtherefore stable microbial activity.