Small-scale photobioreactors (PBRs) in the inoculum stage were designed with internal (red or green) and external white LED light as an initial step of a larger-scale installation aimed at fulfilling the integral biorefinery concept for maximum utilization of microalgal biomass in a multifunctional laboratory. The specific growth rate of Scenedesmus obliquus (Turpin) Kützing biomass for given cultural conditions was analyzed by using MAPLE software. For the determination of total polyphenols, flavonoids, chlorophyll “a” and “b”, carotenoids and lipids, UHPLC-HRMS, ISO-20776/1, ISO-10993-5 and CUPRAC tests were carried out. Under red light growing, a higher content of polyphenols was found, while the green light favoured the flavonoid accumulation in the biomass. Chlorophylls, carotenoids and lipids were in the same order of magnitude in both samples. The dichloromethane extracts obtained from the biomass of each PBR synergistically potentiated at low concentrations (0.01–0.05 mg/mL) the antibacterial activity of penicillin, fluoroquinolones or oregano essential oil against the selected food-borne pathogens (Staphylococcus aureus, Escherichia coli and Salmonella typhimurium) without showing any in vitro cytotoxicity. Both extracts exhibited good cupric ion-reducing antioxidant capacity at concentrations above 0.042–0.08 mg/mL. The UHPLC-HRMS analysis revealed that both extracts contained long chain fatty acids and carotenoids thus explaining their antibacterial and antioxidant potential. The applied engineering approach showed a great potential to modify microalgae metabolism for the synthesis of target compounds by S. obliquus with capacity for the development of health-promoting nutraceuticals for poultry farming.

Escherichia coli (E. coli) is a ubiquitous microorganism with pathogenic and saprophytic clones. The objective of this study was to evaluate the presence, virulence, antibiotic resistance and biofilm formation of E. coli in three industrial farms in Bulgaria, as well as their adjacent sites related to the utilization of manure (feces, wastewater in a separator, lagoons, means of transport, and soils). The isolation of single bacterial cultures was performed via standard procedures with modifications, and E. coli isolates were identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR). The disk diffusion method was used to assess antimicrobial resistance, and PCR was used to detect genes for antibiotic resistance (GAR) (qnr, aac(3), ampC, blaSHV/blaTEM and erm) and virulence genes (stx, stx2all, LT, STa, F4 and eae). The protocol of Stepanović was utilized to measure the biofilm formation of the isolates. A total of 84 isolates from different samples (n = 53) were identified as E. coli. Almost all demonstrated antimicrobial resistance, and most of them demonstrated resistance to multiple antibiotics from different classes. No virulence genes coding the Shiga toxin or enterotoxins or those associated with enteropathogenicity were detected. No GAR from those tested for quinolones, aminoglycosides and macrolides were found. However, all isolates that were resistant to a penicillin-class antibiotic (56) had β-lactamase-producing plasmid genes. All of them had ampC, and 34 of them had blaTEM. A total of 14 isolates formed strongly adherent biofilms. These results in a country where the use of antibiotics for growth promotion and prophylaxis in farms is highly restricted corroborate that the global implemented policy on antibiotics in human medicine and in animal husbandry needs revision.

Yersiniosis is the third most commonly reported foodborne zoonosis in the European Union. Here, we evaluated the prevalence of pathogenic Yersinia enterocolitica among healthy pigs (as a major reservoir) in a slaughterhouse in Bulgaria. A total of 790 tonsils and feces from 601 pigs were examined. Isolation and pathogenicity characterization was carried out by the ISO 10273:2003 protocol and Polymerase Chain Reaction (PCR), detecting the 16S rRNA gene, attachment and invasion locus (ail), Yersinia heat-stable enterotoxin (ystA), and Yersinia adhesion (yadA) genes. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance by the standard disk diffusion method. Of all the pigs tested, 6.7% were positive for Y. enterocolitica. All isolates belonged to Y. enterocolitica bioserotype 4/O:3. ail, and ystA genes were detected in all positive strains (n = 43), while the plasmid Yersinia virulence plasmid (pYV) was detected in 41. High homogeneity was observed among the strains, with all strains susceptible to ceftriaxone, amikacin and ciprofloxacin, and resistant to ampicillin. In conclusion, a low prevalence of Y. enterocolitica 4/O:3 was found in healthy pigs slaughtered in Bulgaria, not underestimating possible contamination of pork as a potential risk to consumer health.

Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin–Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID₅₀ assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15^® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC₅₀) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC₅₀) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.

Antibiotic resistance (ABR) is a significant threat to human and animal health, as well as to the environment. Antibiotic residues, antibiotic-resistant bacteria and antibiotic resistance genes are emerging contaminants of water resources. The present study aimed to assess the prevalence of ABR among waterborne enterococci in an anthropogenic-affected section of the Yantra River (Bulgaria). The susceptibility of 426 strains to 13 antibiotics (ABs) was tested by the disk diffusion method, and the genes encoding resistance by PCR analyses. A total of 39% of isolates were found to be antibiotic-resistant, with 9% mainly being multidrug-resistant to three AB classes. The most common resistance was to erythromycin (19%), tetracycline (18%) and ampicillin (14%), encoded by the ermB, tetM and blaTEM genes. A total of 3% of isolates were ciprofloxacin-resistant and only 1% was resistant to vancomycin or high-level gentamicin. All isolates were susceptible to teicoplanin and linezolid. Spatial variations in ABR levels were found, with the lowest abundance of antibiotic-resistant enterococci occurring in upstream river waters, away from urban areas, and the highest in urban areas. The spread of waterborne antibiotic-resistant enterococci highlights the need for water pollution management, monitoring and control to limit anthropogenic pressures through wastewater discharges and diffuse fecal pollution, and to ensure the ecological well-being of receiving waters.

Coronaviruses are worldwide-distributed RNA viruses with zoonotic potential and the ability to jump from one host species to another, including humans. Even after the COVID-19 pandemic, the search for new, biologically active substances with anti-coronavirus activity continues to be a critical milestone for human health protection. In the framework of a complex engineering strategy, we cultivated the microalgal species Scenedesmus acutus in two different innovative types of flat-plate photobioreactors (PBR1 and K1) for CO₂ utilization and biomass production with special features. Isolated extracts from the microalgal biomass of each one were compared for their anti-coronavirus potential. The design of both PBRs allows a hydrodynamic regime to achieve best fluid flow distribution in their sections, therefore providing the optimal so-called flashing light effect. Of course, this is achieved under well-controlled operational conditions. A strain of beta coronavirus 1 (BCoV, bovine coronavirus) replicated in MDBK cells was used as an in vitro model for the evaluation of the antiviral activity of both extracts. The cell viability, number of survived BCoV particles, and cytopathic effect were evaluated after pre-incubation of the virus with the extracts or direct treatment. The extracts’ samples exhibited evident antiviral activity—extract 1 (from PBR1) in concentrations ≥ 200 µg/mL and extract 2 (from K1) in concentrations ≥150 µg/mL. The ddPCR result revealed significant diminishment of the BCoV particles in samples treated with higher concentrations of the extracts. The phytochemical analysis for certain main groups of compounds (flavonoids, polyphenols, carotenoids, and lipids) showed some differences for both extracts, which could be a possible reason for the observed difference in the antiviral activity. In conclusion, the innovative PBRs are a good platform for studying microalgal growth kinetics by applying different stress conditions from hydrodynamics and mass transfer subsystems. Both extracts showed promising potential for the isolation of metabolites with antiviral activity against BCoV and could be an object for future pharmacological investigations.