Decontamination of Marketed Mullet ( Mugil cephalus ) Infected with Aeromonas hydrophila by Organic Acids

Background and Objective: Aeromonas hydrophila ( A. hydrophila ) is an emerging enteric pathogen that is causing outbreaks in global fish farms, resulting in significant financial losses. Materials and Methods: Using the plate count method, this study investigated the antimicrobial activity of organic acids such as acetic and citric acid on A. hydrophila in mullet ( Mugil cephalus ) at various treatment times (0.5, 1.5, 2.25 and 24 hrs) and temperatures (30±2 E C and 5±2 E C). Results: After a long time (24 hrs) of treatment, the findings of organic acids treatment revealed that acetic acid (5%), citric acid (5-6%) and acetic-citric acids mixes were effective against A. hydrophila at room temperature with a reduction rate of 98.2%, 38.2% and -45.97%, respectively and refrigerator temperature with reduction rate of 97.01%, 15.22% and -28.95%, respectively. At both refrigeration and room temperatures, the acetic acid (5%) showed rising reduction rates that reached almost their highest value after 24 hrs (97.01 and 98.20%, respectively). In addition, citric acid was more effective at room temperature than at refrigerator temperature (0.5, 1.5 and 2.25 hrs). At both temperatures, however, the decrease rate generated by the acetic-citric acid mixture vanished and was almost similar to that of untreated groups. Conclusion: The results of the laboratory investigation suggested that using organic acids (acetic and citric acid) to decontaminate A. hydrophila infection in mullet aquaculture farms is a safe and cost-effective option.


INTRODUCTION
Aeromonas hydrophila as gram-negative non-sporulated bacilli is a facultative anaerobic bacterium commonly existing in aquatic environments where it has emerged as a food-borne pathogen of great significance 1,2 . The infections with A. hydrophila are associated with a lot of syndromes including swelling of tissues, dropsy, red sores, necrosis, ulceration and haemorrhagic septicaemia in tilapia fish 3 . Aeromonas hydrophila has the ability to tolerate frying, grilling and freezing temperatures 4 . Hence, there are extreme public health risks posed by the ready-to-eat fish involving gastroenteritis, wound infection, septicaemia and skin disease, bacteremia, respiratory tract infections, gastroenteritis, urinary tract infection and traveler's diarrhea [5][6][7] .   Temperature  Acetic acid  Citric acid  Acetic-citric acids mixture  Untreated group  Refrigeration T (5±2EC)  20  20  20  20  Room T (30±2EC)  20  20  20  20 acetic-citric acids mixture (G4R). Also, the other four groups were divided into an untreated group (G1F) and treated groups with acetic acid (G2F), citric acid (G3F) and acetic-citric acids mixture (G4F) at refrigerator temperature (5±2EC) ( Table 1).
Experimental examination and parameters studied: All mullets were bathed using sterilized phosphatebuffered saline solution (Difco, Detroit, MI) and then dribble on the aseptic metal mesh. After that, disinfection of mullet was performed by sinking in ethanol (70%) for 5 min, followed by dribbling on sterilized metal mesh in a laminar flow hood. Once thorough occurred, mullets were immersed for 45 min in 1 L of bacteria suspension (with 10 times clockwise and anticlockwise trembling each five min for the full time) 8,15 . Then, the mullet groups (G2R, G3R, G4R) were dipped in baths containing acetic acid, citric acid and acetic-citric acids mixture and kept at RT (30±2EC), while other groups (G2F, G3F, G4F) were dipped in baths containing acetic acid, citric acid, acetic-citric acids mixture and kept at refrigerator temperature (5±2EC). The untreated groups (G1F and G1R) were dipped in a sterile saline solution without any organic acids.
After each specified time (0.5, 1.5, 2.25 and 24 hrs), treated mullets were collected by sterilized forceps and permitted to dry on aseptic material. Ten grams of the musculature of treated mullet were weighed up into 90 mL of sterilized physiological saline (0.85% NaCl) and then homogenization was made in a stomacher under aseptic condition. A serial ten-fold dilution in sterile phosphate buffered saline solution was made from the original homogenate up to 10 6 . Then, 0.1 mL from every dilution was inoculated into MacConkey-ampicillin agar plates (Oxoid, Basingstoke, UK) and the plates were kept in the incubator at 35EC for 24 hrs. On the plate surface, the colonies were counted and the log 10 CFU gG 1 was determined 16 .

Statistical analysis:
The Kruskal-Wallis test (non-parametric ANOVA) was used for the differentiation of the treatment and control means. The p-value was established at<0.05 (SPSS version 20).
Log 10 reduction and reduction percentages were designed by Excel software version 2010: Where, A = log 10 number of viable microbes before treatment B = log 10 number of viable microbes after treatment If the number is negative, this indicates a log 10 increase in number and percentage.

Decontamination of A. hydrophila using organic acids at room temperature:
The efficacy of organic acids on A. hydrophila at room temperature (RT) was evaluated by the reduction in colony counts after different treatment times (0.5, 1.5, 2.25 and 24 hrs) using the plate count method of viable bacterial cells (   The difference between the reduction effects of the two acids on A. hydrophila was significant in which and p<0.0001 Table 3: Effect of organic acids treatments on A. hydrophila count at refrigerator temperature

Decontamination of A. hydrophila using organic acids at refrigeration temperature:
The efficiency of organic acids on A. hydrophila at refrigerator temperature was assessed by the reduction in colony counts after different treatment times (0.5, 1.5, 2.25 and 24 hrs) using the plate count method of viable bacterial cells (Table 3). The acetic acid (97.01%), followed by citric acid (15.22%) and mixtures (-28.95%) were effective against A. hydrophila at refrigerator temperature for a long period of treatment (24 hrs). The citric acid showed the highest reduction rate (15.82%) of A. hydrophila after 0.5 hrs of treatment, while acetic acid revealed the highest reduction rate (12.83, 35.82 and 97.01%) after 1.5, 2.25 and 24 hrs of treatment, respectively. The acetic-citric acid mixture led to a reduction of A. hydrophila to 6.56% after a short duration of treatment only (0.5 hrs) at refrigerator temperature.

Comparison of decontamination at both room and refrigeration temperatures:
The results of organic acids treatment demonstrated that acetic acid (5%) had to descend reducing rates which extended approximately its peak value afterward 24 hrs (97.01 and 98.20%) at both refrigerator and RT, respectively. Furthermore, the effectiveness of acetic acid was highly observed at RT than at refrigerator one. Regarding the influence of citric acid, it was higher in effect than acetic acid only at a short duration (0.5 hrs) at both temperatures. Also, citric acid was more effective at refrigerator temperature than at RT for (0.5, 1.5 and 2.25 hrs) ( Table 2 and 3). The variance between the reduction influence of the two acids on A. hydrophila was statistically significant (p<0.0001).
On the other hand, when acetic and citric acids were mixed, they resulted in a buffering effect between them (weak and strong acids). The acetic-citric acid mixture led to a reduction of A. hydrophila to an undetectable level after 0.5 hrs at both temperatures. The reduction was continued for 1.5 and 2.25 hrs at refrigerator temperature and then the reduction level disappeared at both temperatures after 24 hrs (Table 1 and 2). So, the reduction rate in the mixture of treated samples was nearly equal to that of treated ones.

DISCUSSION
The present results demonstrated that the acetic acid had a reduction rate that reached its peak value after 24 hrs from treatment at either room or refrigerator temperatures. On the other hand, citric acid was more efficient than acetic acid at a short time of 0.5 hrs of treatment at both room and refrigerator temperatures. Regarding temperature, acetic acid and citric acid were more effective at room temperature than refrigerator one after 24 hrs of treatment. The A. hydrophila is not only associated with frequent fish diseases and heavy economic losses, but it also has public health hazards in Egypt 5,12,17 . Thus, for consumers, protection against foodborne pathogens such as A. hydrophila is a global public health concern. Thus, the current investigation studied the decontamination of mullet from A. hydrophila using organic acids. Although the antimicrobial activities of acetic and citric acids have previously been approved 18 , the antimicrobial activity of acetic acid is higher than citric acid 19 . Acetic acid interferes with cytoplasmic membrane structure and membrane proteins for instance the electron transportation is

Limitations:
The present study recommended improving the quality and safety in mullet aquaculture farms through the application of organic acids particularly acetic acid for the decontamination of A.
hydrophila strains. It should be noted that there are some limitations to the present study. Therefore, additional studies are warranted to explore the improvement of growth performance and immune responses of mullets against fish pathogens, especially A. hydrophila infection.

CONCLUSION
In light of the results obtained in the current research, it could be assumed that application of organic

SIGNIFICANCE STATEMENT
The evaluation of the antimicrobial activity of organic acids (acetic and citric acids) on Aeromonas hydrophila that is commonly distributed in aquaculture, particularly marketed mullet (Mugil cephalus) in Egypt. The implication of the results was that the highest reduction rate of acetic acid occurred at 24 hrs of treatment, while citric acid was more efficient at 0.5 hrs of treatment at either room or refrigerator temperatures. Also, acetic acid and citric acid were more effective at room temperature than at refrigerator one after 24 hrs of treatment.