Isolating pathogenic multidrug-resistant Aeromonas hydrophila from diseased fish and assessing the effectiveness of a novel lytic Aeromonas veronii bacteriophage (AVP3) for biocontrol.

The increasing trend of antimicrobial resistance (AMR) pathogens in aquaculture makes it is imperative to find control measures for AMR pathogens causing high economic losses in aquaculture. In the present study, a multidrug resistance (MDR) Aeromonas hydrophila bacterium was isolated from kidney samples of diseased carp originating from a fish farm in Awankot, Rupnagar, Punjab, India. Moribund-infected fish exhibited large irregular hemorrhages on the external body surfaces, exophthalmia and fin-rot-like lesions. Phenotypic characterization using Rimler-Shotts (RS) media showed characteristic yellow color colonies and beta hemolysis on sheep blood agar. Genotyping using species-specific primers for the rpoB and gyrB genes characterized the isolate as A. hydrophila. The Multiple Antibiotic Resistance (MAR) index analysis showed that the isolated A. hydrophila had an MAR score of 0.29 signifying its resistance to more than three antibiotics, which underscores the need of finding treatment methods for MDR A. hydrophila isolates causing disease in aquaculture. Bacteriophages are considered a better eco-friendly alternative to antibiotics because of their inherent properties of not causing drug residues and resistance. Of the 13 phages tested, the Aeromonas veronii phage designated as AVP3, initially isolated against Aeromonas veronii, showed lytic activity against the MDR A. hydrophila isolated from diseased carp in this study. In addition, it also showed the lytic activity against Aeromonas spp. And A. caviae indicating that it had lytic properties against a wide host range within the Aeromonas species. This finding points to the potential efficacy of bacteriophages in mitigating pathogenic infections in aquaculture.

Prevalence and antimicrobial resistance of food safety related Vibrio species in inland saline water shrimp culture farms.

This study evaluated the potential pathogenicity and antimicrobial resistance (AMR) of Vibrio species isolated from inland saline shrimp culture farms. Out of 200 Vibrio isolates obtained from 166 shrimp/water samples, 105 isolates were identified as V. parahaemolyticus and 31 isolates were identified as V. alginolyticus and V. cholerae, respectively. During PCR screening of virulence-associated genes, the presence of the tlh gene was confirmed in 70 and 19 isolates of V. parahaemolyticus and V. alginolyticus, respectively. Besides, 10 isolates of V. parahaemolyticus were also found positive for trh gene. During antibiotic susceptibility testing (AST), very high resistance to cefotaxime (93.0%), amoxiclav (90.3%), ampicillin (88.2%), and ceftazidime (73.7%) was observed in all Vibrio species. Multiple antibiotic resistance (MAR) index values of Vibrio isolates ranged from 0.00 to 0.75, with 90.1% of isolates showing resistance to ≥ 3 antibiotics. The AST and MAR patterns did not significantly vary sample-wise or Vibrio species-wise. During the minimum inhibitory concentration (MIC) testing of various antibiotics against Vibrio isolates, the highest MIC values were recorded for amoxiclav followed by kanamycin. These results indicated that multi-drug resistant Vibrio species could act as the reservoirs of antibiotic resistance genes in the shrimp culture environment. The limited host range of 12 previously isolated V. parahaemolyticus phages against V. parahaemolyticus isolates from this study indicated that multiple strains of V. parahaemolyticus were prevalent in inland saline shrimp culture farms. The findings of the current study emphasize that routine monitoring of emerging aquaculture areas is critical for AMR pathogen risk assessment.

Gelation characteristics of partially purified myofibrillar proteins extracted from commercially harvested Indian mackerel and threadfin bream.

Gelling behaviors of partially purified myofibrillar proteins (PPMP) extracted from Indian mackerel (IM) and threadfin bream (TB) as a function of heating temperatures (20-75°C) were comparatively studied. PPMP obtained from IM (IM-MP) showed lower turbidity and surface hydrophobicity as compared to those extracted from TB (TB-MP). Moreover, lower disulfide bond content was noticed in IM-MP (7.7-9.46 mol/106  g protein) as compared to TB-MP (10.99-13.95 mol/106  g protein) during the heating process. There was no major difference in the amino acid profile noticed between PPMP from both the species, except lysine and glutamine contents, which were higher in TB-MP. Structural analysis, FTIR spectra, amide I band, and fluorescence intensity substantiated those changes. The protein pattern also revealed autolysis of IM-MP. The transmission analysis also showed lower aggregation and crosslinking ability of IM-MP than TB-MP. Therefore, poorer gelling behavior of IM-MP reconfirmed the inferior gel property of surimi gel from IM to gel from TB. Potential development is still required for the improvement of the gel properties of dark-fleshed fish surimi such as IM. PRACTICAL APPLICATION: Indian mackerel (IM) is an abundant and widely captured fish species. Due to overexploitation of lean fish, pelagic fish could be explored as a potential raw material for surimi production. However, poor gelling properties of IM limit its use in the surimi industry. This study provides an insight into the gelling behavior of myofibrillar proteins from IM during the gelation process in comparison with the lean fish (threadfin bream). Overall, structural and rheological changes of myofibrillar proteins play a role in gelation, thus affecting gel properties between two species. Further improvement of the gel of IM is still required.

Ethanolic Extract of Duea Ching Fruit: Extraction, Characterization and Its Effect on the Properties and Storage Stability of Sardine Surimi Gel.

The quality of surimi gel can be improved using protein cross-linkers, especially from plant extracts. Apart from the presence of phenolic compounds, Duea ching fruit is rich in calcium, which can activate indigenous transglutaminase or form the salt bridge between protein chains. Its extract can serve as a potential additive for surimi. The effect of different media for the extraction of Duea ching was studied and the use of the extract in sardine surimi gel was also investigated. The Duea ching fruit extract (DCE) was prepared using distilled water and ethanol (EtOH) at varying concentrations. The DCE prepared using 60% EtOH (DCE-60) had the highest antioxidant activity and total phenolic content. When DCE-60 (0-0.125%; w/w) was added to the sardine surimi gel, the breaking force (BF), deformation (DF) and water holding capacity (WHC) of the gel upsurged and the highest values were attained with the 0.05% DCE-60 addition (p < 0.05). However, the whiteness of the gel decreased when DCE-60 levels were augmented. The gel containing 0.05% DCE-60, namely D60-0.05, showed a denser network and had a higher overall likeness score than the control. When the D60-0.05 gel was packed in air, under vacuum or modified atmospheric packaging and stored at 4 °C, BF, DF, WHC and whiteness gradually decreased throughout 12 days of storage. However, the D60-0.05 gel sample showed lower deterioration than the control, regardless of the packaging. Moreover, the gel packaged under vacuum conditions showed the lowest reduction in properties throughout the storage than those packaged with another two conditions. Thus, the incorporation of 0.05% DCE-60 could improve the properties of sardine surimi gel and the deterioration of the resulting gel was retarded when stored at 4 °C under vacuum packaging conditions.

Impact of Ethanolic Thai Indigenous Leaf Extracts on Melanosis Prevention and Shelf-Life Extension of Refrigerated Pacific White Shrimp.

Shrimp has been known for its delicacy, but it undergoes rapid deterioration induced by biochemical and microbiological reactions. Melanosis is a major cause of discoloration associated with consumer rejection. All ethanolic extracts from different leaves including soursop, noni, and Jik leaves were dechlorophyllized via the "Green" sedimentation method before being used. The inhibitory activity against polyphenoloxidase (PPO) from Pacific white shrimp (Litopeneous vannamei) and the copper-chelating properties of varying extracts were compared. Soursop leaf extract (SLE) showed higher PPO inhibitory activity and copper-chelating ability than others (p < 0.05). Based on LC-MS, aempferol-3-O-rutinoside was identified as the most abundant compound, followed by catechin and neocholorigenic acid. The efficacy of SLE at different levels (0.25-1%) for inhibiting melanosis and preserving the quality of Pacific white shrimp was evaluated during refrigerated storage at 4 °C for 12 days in comparison with that of a 1.25% sodium metabisulfite (SMS)-treated sample. SLE at a level of 1% effectively retarded melanosis and bacterial growth, in which the total viable count did not exceed the microbial limit within 12 days. In addition, 1% SLE treatment impeded autolysis, reduced protein degradation and decomposition, and minimized lipid oxidation, as witnessed by the lower increases in pH, TVB-N, and TBARS values. Sensory evaluation indicated higher likeness scores and overall acceptability for SLE-1% and SMS-1.25% shrimps than those of the control and other samples. Therefore, SLE could be used as a natural alternative that effectively lowered the melanosis and quality loss of shrimp during refrigerated storage.

Vibrio parahaemolyticus Isolates from Asian Green Mussel: Molecular Characteristics, Virulence and Their Inhibition by Chitooligosaccharide-Tea Polyphenol Conjugates.

Fifty isolates of Vibrio parahaemolyticus were tested for pathogenicity, biofilm formation, motility, and antibiotic resistance. Antimicrobial activity of chitooligosaccharide (COS)-tea polyphenol conjugates against all isolates was also studied. Forty-three isolates were randomly selected from 520 isolates from Asian green mussel (Perna viridis) grown on CHROMagarTM Vibrio agar plate. Six isolates were acquired from stool specimens of diarrhea patients. One laboratory strain was V. parahaemolyticus PSU.SCB.16S.14. Among all isolates tested, 12% of V. parahaemolyticus carried the tdh+trh- gene and were positive toward Kanagawa phenomenon test. All of V. parahaemolyticus isolates could produce biofilm and showed relatively strong motile ability. When COS-catechin conjugate (COS-CAT) and COS-epigallocatechin-3-gallate conjugate (COS-EGCG) were examined for their inhibitory effect against V. parahaemolyticus, the former showed the higher bactericidal activity with the MBC value of 1.024 mg/mL against both pathogenic and non-pathogenic strains. Most of the representative Asian green mussel V. parahaemolyticus isolates exhibited high sensitivity to all antibiotics, whereas one isolate showed the intermediate resistance to cefuroxime. However, the representative clinical isolates were highly resistant to nine types of antibiotics and had multiple antibiotic resistance (MAR) index of 0.64. Thus, COS-CAT could be used as potential antimicrobial agent for controlling V. parahaemolyticus-causing disease in Asian green mussel.