A challenge test on Pseudomonas spp. as spoiling microorganism in fish fillets.

Fish fillets are highly susceptible to spoilage, with Pseudomonas spp. bacteria being among the main culprits. To maintain products' quality and safety, it is important to control the load of these microorganisms and understand their growth potential in fish fillets. However, setting up challenge tests might be hard due to the difficulty of differentiating intentionally inoculated bacteria from those already present on the fillets. To overcome this obstacle, a pilot study using Pseudomonas aeruginosa, a clinically significant bacterial species that is rare in food, was conducted. Vacuum-packed Northern cod, salmon, and plaice fish fillets were experimentally inoculated and subjected to trials at both refrigeration (4 °C) and thermal abuse temperatures (from +4 °C to +6 °C and then to +8 °C). The results showed that the growth potential of Pseudomonas aeruginosa in all the fish fillets was less than 0.5 Log10 CFU/g. This confirms that vacuum packaging could reduce the multiplication of Pseudomonas spp. in the fish fillets and underlines as it is crucial to have fish fillets containing initial loads of Pseudomonas between 104-105 CFU/g or lower at the beginning of the shelf life in order to control the deterioration rate of the product. This study provides a basis for developing further challenge tests for Pseudomonas spp. in the fish industry and highlights the importance of controlling initial loads of Pseudomonas to prevent product deterioration during the shelf life.

The sensitivity of Daphnia magna and Daphnia curvirostris to 10 veterinary antibacterials and to some of their binary mixtures.

Aim of this study was to evaluate the suitability of Daphnia curvirostris for the acute toxicity test usually performed on Daphnia magna, and to compare the sensitivity of the two species toward 10 antibacterials [enrofloxacin (EFX), ciprofloxacin(CPX), sulfaguanidine (SGD), sulfadiazine (SDZ), sulfamethazine (SMZ), sulfaquinoxaline (SQO), sulfaclozine (SCZ), sulfamerazine (SMA), sulfadimethoxine (SDM) and trimethoprim (TMP)] and some of their binary mixtures. Furthermore, a tentative prolonged-toxicity test (lasting 13d) was settled up in order to evidence toxic responses with drug concentrations that were uneffective in the classic 48h immobilization test. Results showed that D. curvirostris was more sensitive than D. magna to the majority of compounds (6 out of 10). Lowest 48h EC50s were obtained with EFX (4.3mgL(-1) in D. curvirostris) and SGD (6.2mgL(-1) in D. magna). The toxicity of paired compounds was always concentration-additive or less than concentration-additive. In the prolonged-toxicity test mortality and/or reproduction inhibition were constantly observed. It was concluded that: (1) D. curvirostris could be a suitable model for the evaluation of acute toxicity of antibacterials since its sensitivity was generally greater than that of D. magna; (2) the toxicity of EFX and SGD should be given special attention as the two compounds, in the prolonged test, showed to be active at concentrations of 0.9mgL(-1) and 2.5mgL(-1), respectively; (3) the concentration addition is usually a reasonable worst case estimation of the environmental impact of antibacterial mixtures.

A monitoring of chemical contaminants in waters used for field irrigation and livestock watering in the Veneto region (Italy), using bioassays as a screening tool.

In this study, 50 livestock watering sources (ground water) and 50 field irrigation sources (surface water) from various industrialised areas of the Veneto region were monitored for chemical contaminants. From each site, four water samples (one in each season) were collected during the period from summer 2009 through to spring 2010. Surface water samples and ground water samples were first screened for toxicity using the growth inhibition test on Pseudokirchneriella subcapitata and the immobilisation test on Daphnia magna, respectively. Then, based on the results of these toxicity tests, 28 ground water samples and 26 surface water samples were submitted to chemical analysis for various contaminants (insecticides/acaricides, fungicides, herbicides, metals and anions) by means of UPLC-MS(n) HPLC-MS(n), AAS and IEC. With the exception of one surface water sample where the total pesticides concentration was greater than 4 µg L(-1), positive samples (51.9 %) showed only traces (nanograms per liter) of pesticides. Metals were generally under the detection limit. High concentrations of chlorines (up to 692 mg L(-1)) were found in some ground water samples while some surface water samples showed an excess of nitrites (up to 336 mg L(-1)). Detected levels of contamination were generally too low to justify the toxicity recorded in bioassays, especially in the case of surface water samples, and analytical results painted quite a reassuring picture, while tests on P. subcapitata showed a strong growth inhibition activity. It was concluded that, from an ecotoxicological point of view, surface waters used for field irrigation in the Veneto region cannot be considered safe.

Approved medication of water with enrofloxacin to treat turkey colibacillosis: assessment of efficacy using a PK/PD approach.

The efficacy of administering enrofloxacin at 10mg/kg in medicated water to turkeys was evaluated by applying a PK/PD approach to the kinetic parameters obtained after oral pulsed administration and to the MIC values of avian pathogenic Escherichia coli (APEC) strains isolated from commercial turkey flocks. The kinetic parameters of enrofloxacin were evaluated in 10 healthy and 10 colisepticemic turkeys that received the drug dissolved in medicated water at 89 µg/mL and 71 µg/mL, respectively, for 10h/day for 5 days. Blood samples were collected for 24h from all turkeys on the last day of treatment, and the serum was analysed by HPLC with fluorimetric detection. The mean AUC (7374.53±1067.64 h ng/mL and 7656.95±1460.61 h ng/mL) and C(max) values (673.09±186.18 ng/mL and 543.50±68.75 ng/mL) obtained for healthy and sick turkeys were not significantly different. High-level resistance was observed in 30.3% of strains, 40.5% exhibited intermediate resistance, and only 29.2% were susceptible; the MIC(50) and MIC(90) values were 1mg/L and 32 mg/L, respectively. The PK/PD parameters C(max)/MIC(50) (0.67 and 0.54 for healthy and sick turkeys, respectively) and AUC/MIC(50) (7.37 and 7.66) were lower than the efficacy breakpoints reported for fluoroquinolones. These results indicate that authorised dosage of enrofloxacin used in pulsed medicated water administration could be ineffective against more than the 70% of circulating APEC strains, indicating the need to test the drug susceptibility of APEC prior to administering the drug and adopting a more convenient medication schedule for mass treatment.

Sublethal effects of trimethoprim on four freshwater organisms.

Sublethal effects of trimethoprim (TMP) were evaluated in four freshwater organisms: Pseudokirchneriella subcapitata and Lemna minor (growth inhibition), Daphnia magna (reproduction and growth inhibition) and Poecilia reticulata (swimming activity inhibition). Cytochrome P4501A induction was also evaluated in P. reticulata. TMP showed varying levels of toxicity in the four test performed, with NOEC for the various endpoints in the range of 3.12-25 mg L(-1). The compound was active on P. reticulata at concentration ≥ 50 mg L(-1) causing inhibition of swimming activity. In the same organism an induction of CYP1A protein, mainly in kidney, gills and intestine, was also detected. L. minor was more sensitive than unicellular algae to TMP, with a NOEC of 12.5 mg L(-1). The lowest NOEC (3.12 mg L(-1)) was obtained in D. magna reproduction test and then a Risk Quotient of <0.03 was calculated by comparing the PNEC (31.2 µg L) and the TMP concentrations usually detected in freshwater (<1 µg L(-1)). However, based on recently reported data, it was concluded that while TMP concentrations normally detected in surface water are below those able to evoke appreciable biological effects in the various aquatic organisms, TMP concentrations in aquaculture and hospital effluents can be one to three orders of magnitude higher. Furthermore, the co-occurrence and additive effects of other antifolic agents should be taken into account for a cautious risk assessment of the drug.

Evaluation of the aquatic toxicity of two veterinary sulfonamides using five test organisms.

The aquatic toxicity of sulfaquinoxaline (SQO) and sulfaguanidine (SGD) was evaluated on the following test organisms: Daphnia magna (reproduction test), Pseudokirchneriella subcapitata, Scenedesmus dimorphus, Synecococcus leopoliensis (algal growth inhibition test) and Lemna gibba (duckweed growth inhibition test). Furthermore, the additivity of the two compounds was measured on D. magna (acute immobilisation test) and P. subcapitata (algal growth inhibition test) using the isobologram method. Results show that SQO and SGD are more toxic to green algae and daphnids, respectively, than other veterinary sulfonamides (SAs) and that their mixtures have a less then additive interaction. Taking into account the highest concentrations detected so far in surface waters for SQO (0.112 µg L(-1)) and for SGD (0.145 µg L(-1)) and the lowest NOECs obtained with the five test organisms, divided by an assessment factor of 10, the following PNECs and risk quotients (RQs) were calculated. SQO: PNEC 2 µg L(-1); RQ 0.056. SGD: PNEC 39.5 µg L(-1); RQ 0.004. Consequently, at the concentrations actually detected in the aquatic environment, the two SAs alone should not harm the freshwater organisms. However, it seems advisable, for veterinary mass treatments, the use of other SAs that have a lesser impact on the aquatic environment. Furthermore, considering the high probability of having complex mixtures of different SAs residues in water, each individual contamination should be evaluated by applying to the SAs mixtures the conservative criteria of additivity.