Antimicrobial Resistance in Aquaculture: Risk Mitigation within the One Health Context.

The application of antimicrobials in aquaculture primarily aims to prevent and treat bacterial infections in fish, but their inappropriate use may result in the emergence of zoonotic antibiotic-resistant bacteria and the subsequent transmission of resistant strains to humans via food consumption. The aquatic environment serves as a potential reservoir for resistant bacteria, providing an ideal breeding ground for development of antimicrobial resistance (AMR). The mutual inter-connection of intensive fish-farming systems with terrestrial environments, the food processing industry and human population creates pathways for the transmission of resistant bacteria, exacerbating the problem further. The aim of this study was to provide an overview of the most effective and available risk mitigation strategies to tackle AMR in aquaculture, based on the One Health (OH) concept. The stringent antimicrobial use guidelines, promoting disease control methods like enhanced farm biosecurity measures and vaccinations, alternatives to antibiotics (ABs) (prebiotics, probiotics, immunostimulants, essential oils (EOs), peptides and phage therapy), feeding practices, genetics, monitoring water quality, and improving wastewater treatment, rather than applying excessive use of antimicrobials, can effectively prevent the development of AMR and release of resistant bacteria into the environment and food. The contribution of the environment to AMR development traditionally receives less attention, and, therefore, environmental aspects should be included more prominently in OH efforts to predict, detect and prevent the risks to health. This is of particular importance for low and middle-income countries with a lack of integration of the national AMR action plans (NAPs) with the aquaculture-producing environment. Integrated control of AMR in fisheries based on the OH approach can contribute to substantial decrease in resistance, and such is the case in Asia, where in aquaculture, the percentage of antimicrobial compounds with resistance exceeding 50% (P50) decreased from 52% to 22% within the period of the previous two decades.

Determination of antilisterial effect of some microbial isolates from traditional zlatar cheese during the fermentation of soft white cheese.

The aim of this study was to select autochthonous lactic acid bacteria (LAB) isolates with antilisterial activity from Zlatar cheese and to evaluate the ability of selected LAB to control Listeria monocytogenes growth during soft white cheese production. The genotype characterization of isolated LAB (n = 93) was done using PCR method by 16S rRNA sequencing. In this way, the following isolates were detected: Lactococcus lactis ssp. lactis (40 isolates), Enterococcus faecalis (30), Lactobacillus plantarum (12), Leuconostoc mesenteroides ssp. mesenteroides (3) Lactobacillus garvieae (3), Lactobacillus curvatus (2), Lactobacillus casei (1), Enterococcus faecium (1) and Staphylococcus hominis (1). Each isolated LAB was tested for bacteriocin-producing ability. It was determined that two LAB isolates had bactericidal properties: Lactococcus lactis ssp. lactis SRB/ZS/094 and Enterococcus faecalis SRB/ZS/090. Semi-purified of enterococcal bacteriocin (enterocin) was isolated using precipitation procedures with ammonium sulphate. Its properties were determined (strength and range of activities). Isolated enterocin and bacteriocin-producing Lactococcus strain showed significant antimicribial activity against Listeria monocytogenes, but still the inhibition activity of Staphylococcus aurues and Escherichia coli was not detected. Based on the obtained laboratory results, in the second phase of the research, the antilisterial effect of bacteriocin isolated from Enterococcus faecalis SRB/ZS/090 and cells Lactococcus lactis ssp. lactis SRB/ZS/094 were determined, that are added as additives in the production of soft white cheese through five variants. Cheese supplemented with enterocin (E2) had the lowest aerobic mesophilic bacteria count, indicating that enterocin (E2) play an important role for bio-preservation.

Seroprevalence of hepatitis E in pigs and wild boars in the region of the city Belgrade.

INTRODUCTION: Hepatitis E is considered an emerging human viral disease with many evidences of zoonotic nature of disease, and swine are the main reservoir of HEV. The aim of this study was to determine HEV seroprevalence in commercial pig farms, backyard pigs, slaughtered pigs and wild boars in the region of the city Belgrade. METHODOLOGY: A total of 405 sera samples: 150 samples from 3 commercial pig farms, 70 samples from backyard pigs, 119 samples from slaughtered pigs and 66 samples from wild boars of the region of the city Belgrade, Serbia were analysed by commercial ELISA test. RESULTS: The overall HEV seroprevalence in 3 commercial pig farms was 55.33% (83/150). All tested farms (farm A, B and C) were positive on the presence of anti-HEV antibodies, respectively 58% (29/50), 54% (27/50) and 54% (27/50). From 70 tested backyard pigs, 75.71% (53/70) were tested seropositive. In total, 26 backyard pig holidngs were confirmed as positive to anti-HEV antibodies (81.25%). At slaughterhouse, 25% (8/32) weaned piglets and 20.69% (18/87) fattening pigs were tested positive on anti-HEV antibodies. Overall HEV seroprevalence in tested wild boar population was 52.25% (36/66). CONCLUSIONS: Detected very high seroprevalence of anti-HEV antibodies indicated an active circulation of HEV, being enzootic in the swine population, and wild boars, as well, in the region of the city Belgrade.

Subchronic exposure to static magnetic field differently affects zinc and copper content in murine organs.

PURPOSE: Static magnetic fields (SMF) have been widely used in research, medicine and industry. Since zinc and copper play an important role in biological systems, we studied the effects of the subchronic continuous SMF exposure on their distribution in murine tissues. MATERIALS AND METHODS: For 30 days, mice were exposed to inhomogeneous, vertical, downward or upward oriented SMF of 1 mT averaged intensity with spatial gradient in vertical direction. RESULTS: SMF decreased the amount of copper and zinc in liver. In brain, zinc levels were increased and copper levels were decreased. In spleen, zinc content was reduced, while copper amount remained unchanged. CONCLUSIONS: Subchronic exposure to SMF differently affected copper and zinc content in examined organs, and the changes were more pronounced for the downward oriented field. The outcome could be attributed to the protective, rather than the harmful effect of SMF.

Antioxidant and antimicrobial activity of Kitaibelia vitifolia extract as alternative to the added nitrite in fermented dry sausage.

Fermented dry sausages (FDS) without nitrite added, fortified with bioactive phenol and flavonoid compounds originating from the ethanol extract of Kitaibelia vitifolia were food matrix for investigation of its antioxidant and antimicrobial potency. These activities were researched in order to improve the sausages' shelf-life, safety, and provide health benefits to consumers as well. The oxidative stability of the FDS, containing two different levels of natural preservative, was evaluated using five different contemporary methods for antioxidative activity. The activity was tested on the 20th day of the refrigerated storage. Minimum inhibitory concentrations of the sausage extract were determined against six microorganisms, using a micro dilution method. Determined optimal effective concentration of dissolved K. vitifolia extract (12.5 g/kg of meat dough) revealed strong antioxidant activity, and moderate antimicrobial activity against Escherichia coli (minimum inhibitory concentrations=15.625 µg/mL). The modified sausages had typical chemical-physical characteristics of FDS, controlled on 0, 13, 26 d of ripening and 20, 40 and 60 d of storage.

Hematological parameters' changes in mice subchronically exposed to static magnetic fields of different orientations.

Static magnetic fields (SMFs) are time independent fields whose intensity can be spatially dependent. This study investigates influence of subchronic continuous exposure to upward and downward directed SMF on hematological parameters and spleen cellularity in mice. The experiment is performed on the Northern hemisphere; consequently, the vertical component of geomagnetic field is directed downward. Male, Swiss-Webster, 6 weeks old mice were exposed to the vertically declining SMF. Mice were divided in three groups and continuously exposed or not exposed for 28 days to the SMF characterized by the averaged field of 16 mT and averaged field gradient of 10 mT/cm. Differently oriented SMF did not alter hemoglobin and hematocrit content among the groups. However, the groups exposed to the upward and downward fields had statistically significant higher levels of serum transferrin compared to the control. Moreover, spleen cellularity in animals in the downward group was significantly higher compared to the upward and control group. In addition, spleen lymphocytes in both of the exposed groups were significantly higher than in the control group. In contrast, spleen granulocytes in the exposed groups were significantly lower than in the control group. Significant decrease was also observed in brain and liver iron content with concomitant increase of iron in serum and spleen in exposed animals. Subchronic continuous exposure to 16 mT SMF caused lymphocyte and granulocyte redistribution between spleen and blood. This distribution is typical for stress induced hematological changes. These results suggest that observed changes were not due to an unspecific stress response, but that they were rather caused by specific adaptation to subchronic SMF exposure.

Evaluation and validation of two different chromatographic methods (HPLC and LC-MS/MS) for the determination and confirmation of ochratoxin A in pig tissues.

This study was undertaken to compare two different analytical methods for the determination and confirmation of ochratoxin A (OTA) in blood serum, kidney and liver of pigs. Sample clean-up was based on liquid-liquid phase extraction. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with fluorescence detection (FL) or electro spray ionization (ESI+) tandem mass spectrometry (MS-MS). Comparative method evaluation was based on the investigation of 90 samples of blood serum, kidney and liver per animal originating from different regions of Serbia. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with (LC-MS-MS) liquid chromatography electro spray tandem mass spectro metry detection offered comparable good results at the sub-ppb concentration level. The results indicate that the liquid chromatography electro spray tandem mass spectrometric (LC-MS/MS) method was more specific and sensitive for the analysis and confirmation of ochratoxin A in pig tissues then high pressure liquid chromatography (HPLC) method after methylation of OTA.

Quantifying nonthermal inactivation of Listeria monocytogenes in European fermented sausages using bacteriocinogenic lactic acid bacteria or their bacteriocins: a case study for risk assessment.

Listeria monocytogenes NCTC10527 was examined with respect to its nonthermal inactivation kinetics in fermented sausages from four European countries: Serbia-Montenegro, Hungary, Croatia, and Bosnia-Herzegovina. The goal was to quantify the effect of fermentation and ripening conditions on L. monocytogenes with the simultaneous presence or absence of bacteriocin-producing lactic acid bacteria (i.e., Lactobacillus sakei). Different models were used to fit the experimental data and to calculate the kinetic parameters. The best model was chosen based on statistical comparisons. The Baranyi model was selected because it fitted the data better in most (73%) of the cases. The results from the challenge experiments and the subsequent statistical analysis indicated that relative to the control condition the addition of L. sakei strains reduced the time required for a 4-log reduction of L. monocytogenes (t(4D)). In contrast, the addition of the bacteriocins mesenterocin Y and sakacin P decreased the t(4D) values for only the Serbian product. A case study for risk assessment also was conducted. The data of initial population and t(4D) collected from all countries were described by a single distribution function. Storage temperature, packaging method, pH, and water activity of the final products were used to calculate the inactivation of L. monocytogenes that might occur during storage of the final product (U.S. Department of Agriculture Pathogen Modeling Program version 7.0). Simulation results indicated that the addition of L. sakei strains significantly decreased the simulated L. monocytogenes concentration of ready-to-eat fermented sausages at the time of consumption.