Vibriosis occurs in cultured and wild marine fish in salt or brackish water, particularly in shallow waters during late summer. It was originally believed that scavenger fish feeding around the farms were the natural reservoir of V. anguillarum, and contact between fish seems to be an important factor for the spread of this pathogen. However, there is evidence that V. anguillarum is normally present in the intestinal microflora and food of cultured and wild healthy fish. The temperature and quality of the water, the virulence of the V. anguillarum strain and stress on the fish are important elements influencing the onset of disease outbreaks.

Antibiotic resistance in Aeromonas salmonicida

In the 1950s, furunculosis in salmonid hatcheries in the USA was treated with sulphonamides, and an early report on acquired sulphonamide resistance in Aeromonas salmonicida comes from Leetown, West Virginia, with 36 out of 47 isolates from brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta) found to be resistant in 1955.³ In this report, the devastating effect of acquired sulphonamide resistance in the treatment of furunculosis was demonstrated by clinical trials. The resistant strain has since been shown to contain transferable resistance to both sulphonamides and tetracyclines.^5,34

In France in 1971, 104 isolates of A. salmonicida were reported to be 100% resistant to sulphonamides while only 11.5% of the isolates were resistant to the antibiotics tetracycline, streptomycin and/or chloramphenicol.³⁵ Half of the drug-resistant Japanese A. salmonicida from salmonids in the 1960s had R plasmids hosting the drug resistance genes. Sulphonamides, tetracyclines, chloramphenicol and streptomycin were ineffective, although streptomycin was not used in the control of fish diseases.⁵

A study of drug resistance in A. salmonicida isolated from the intestinal tract of diseased pond-reared Amago (O. rhodurus macrostomus) and Yamame (O. masou ishikawae) was conducted in the early 1970s.³⁶ Fish ponds from the Nagano, Gifu, Shiga and Tokyo districts of Japan were studied, and all ponds (24) were supplied with fresh flowing water from springs or mountain streams. The drugs used for treating diseased fish in the ponds were chloramphenicol, sulphonamides, and to a minor extent tetracyclines. All the A. salmonicida isolates (20) found on media with chloramphenicol contained transferable R plasmids conferring resistance towards sulphonamides, streptomycin and chloramphenicol. A large portion of the Aeromonas liquefaciens(hydrophila) strains isolated from the pond-reared fish in this study also contained a transferable R plasmid conferring resistance to sulphonamides, streptomycin and chloramphenicol.

The transferable R plasmids with resistance towards sulphonamides, streptomycin and chloramphenicol isolated from both A. salmonicida and A. liquefaciens (hydrophila) were found to be of the same size and same incompatibility group.³⁷No exchange of salmonid fish between different areas had occurred because this was restricted in an attempt to reduce transmittance of various diseases. In spite of that, the same R plasmid was detected in all fish farms in the four different districts in Japan, indicating that the plasmid occurred naturally in the environmental flora of the freshwater systems.

A. salmonicida isolated during 1979–81 from fish farms in eight districts in Japan, including farms from three of the four districts studied by Aoki et al. in 1972,³⁶ were investigated for antibiotic resistance.³⁹ Of 129 isolates, only five were found to be susceptible to all antibiotic drugs tested. The other isolates were found to be resistant to up to six drugs. In particular, resistance was detected against quinolones and nitrofurans. Only two of the 124 resistant isolates studied were found to transfer drug resistance by R plasmids. These two isolates hosted an R plasmid (29 MDa) conferring resistance against sulphonamides, chloramphenicol and streptomycin. Variants of this IncU R plasmid have been found in Aeromonas bacteria all over the world.^18,41,50

In 1989 in Atlantic salmon (Salmo salar) farms on the western coast of Norway, it was found that infections caused by atypical Aeromonas salmonicida could not be controlled with the quinolone drug oxolinic acid because of chromosomal quinolone resistance. In addition, these strains were found to carry transferable resistance towards sulphonamides, trimethoprim and tetracyclines. An R plasmid of 25 MDa (IncU) was found to be identical to an R plasmid isolated from salmon with furunculosis caused by A. salmonicida ss salmonicida in farms in the same area in 1991.¹³

In Scottish Atlantic salmon farms, A. salmonicida isolates from 229 outbreaks of furunculosis in 44 units at 34 locations during 1988–90 were investigated for antibiotic resistance. Among 304 isolates, 55% were found to be resistant to tetracyclines, 37% resistant to oxolinic acid, 31% resistant to sulphonamides and 10% resistant to a combination of sulphonamides and trimethoprim.⁵¹ Among 40 oxytetracycline resistant isolates, 11 of the isolates transferred an R plasmid which encoded resistance to oxytetracycline and/or sulphonamides, trimethoprim and streptomycin to E. coli. This R plasmid was shown to be an IncU plasmid of the same type as found in Japan and Norway.⁵²

In a study of 29 oxytetracycline resistant A. salmonicida isolates from Scotland, 19 transferred their tetracycline resistance to E. coli.⁵² The Norwegian pRAS1 isolated from A. salmonicida were included in this study and restriction digests showed obvious similarity to the Scottish R plasmids, which were of similar size even if the featured resistance patterns were not identical. In another study, R plasmids featuring oxytetracycline resistance from mesophilic, motile Aeromonas spp. isolated from freshwater in a fish hatchery and from hospital sewage in England, were studied molecularly.⁴³ Six of 91 isolates from the fish hatchery water and 11 of 72 isolates from hospital sewage water were found to transfer oxytetracycline resistance. Seven of the 11 R plasmids from Aeromonas spp. from sewage were shown to belong to the IncU group.

In 23 drug-resistant A. salmonicida isolates from 13 different freshwater rainbow trout farms in Denmark, only three isolates transferred their R plasmid to E. coli. These three isolates transferred an R plasmid of 150 kb with resistance to oxytetracycline, sulphonamides, trimethoprim and streptomycin.⁴⁶ In addition, three oxytetracycline and sulphonamide resistant isolates of A. salmonicida from Canada and one oxytetracycline resistant isolate from USA, included as controls in the study, transferred R plasmids of 140–160 kb. Five A. salmonicida isolates from the Faroe Islands resistant towards oxytetracycline, sulphonamides, trimethoprim and streptomycin were included in the study and they transferred a 50 kb (probably IncU) plasmid to E. coli encoding resistance to all their drug resistance features except streptomycin.

IncU R plasmids were detected in drug-resistant atypical A. salmonicida isolates from farmed and wild fish on the Northeastern coasts of USA and Canada.⁵³

The occurrence of a transferable R plasmid belonging to the IncUgroup in A. salmonicida isolates from various parts of the world in both freshwater systems and in the marine environment is remarkable. This plasmid was found to be identical in all the sampled locations except for the drug resistance region. The IncU plasmid is probably particularly successful at establishing in A. salmonicida. However, the IncU plasmid is promiscuous and was found in Aeromonas hydrophila (see later), in atypical A. salmonicida on the coast of Norway, on the northern part of the east coast of North America and in human enterobacteria in Eastern Europe. Hedges et al.⁴⁰ suggested that the homogenic IncU plasmids are characteristic of aeromonads but do occasionally transfer to and establish in other genera. The plasmids may acquire some of the genetic factors linked to drug resistance in the gene pools of these bacteria and subsequently bring these back to aeromonads. However, one should not rule out the opposite possibility, that these IncU plasmids may be bringing antibiotic resistance factors from aquatic environments into our antibiotic consuming environments.

The use of quinolones, oxolinic acid and flumequine in the control of bacterial infections in farmed fish from the 1980s resulted in the occurrence of A. salmonicida with reduced susceptibility to quinolones.^54,55 Mutations in the gyrase A gene, gyrA, were found to be responsible for elevated MIC values against quinolones in clinical strains of A. salmonicida.⁵⁶ A study of 12 clinical A. salmonicida isolates from French marine fish farms in the period 1998 to 2000 showed that all quinolone resistant isolates carried a point mutation in gyrA.⁵⁷

Quinolone resistant A. salmonicida isolates have been found to have changes in the outer membrane profile with loss of a 38.5 kDa protein and occurrence of a 37 kDa protein, probably linked to loss of porine function resulting in multiple low-level antibiotic resistance.^58,59

In addition to amino acid substitutions in the gyrase A enzyme and porine changes in the outer membranes, it was indicated that an efflux mechanism might be involved in quinolone resistance in strains of A. salmonicida isolated from French marine fish farms.⁵⁷

Quinolones, oxolinic acid and flumequine, were used extensively in fish farming to control infections caused by A. salmonicidafrom the late 1980s until effective vaccines containing oil adjuvants were introduced in the early 1990s. During that period, high MIC values for quinolones developed in isolates of A. salmonicida in all areas where furunculosis was endemic in salmon farming, resulting in repeated treatments of diseased fish during the summer months with only weeks between each treatment. In Norway, the first resistance to quinolones were discovered in 1989, about one year after fish farmers started using oxolinic acid to control furunculosis.⁵⁵ It was found that 36% of the isolates were resistant to one or more of the drugs tested, with 30% resistant to quinolones as the most widespread feature. After many years of effective vaccination, there is minimal information on the occurrence of antimicrobial resistance in strains of A. salmonicida in general. It has been shown that A. salmonicida share similar genetic tools worldwide when drug resistance evolves. 

What is ammonia? and why the rise and fall of  aquaculture industries highly dependable on effective control of ammonia.

Before I go into detail of those Nitrifying and denitrifying bacteria, let me give a short brief introduction of nitrogen cycle and water nitrification. If u want to be successful in aquaculture farming, you must understanding  a very importance concept – nitrogen cycle.

 

Ammonification ——>Nitrification———>  Denitrification.

Ammonification. While traveling through sewer pipes, the majority of the nitrogen contained in raw sewage (urea and fecal material) is converted from organic nitrogen to ammonia through a process called hydrolysis, accomplished by putrefying bacteria and fungus. Technically, in the majority of situations, more ammonium than ammonia is created during ammonification.The actual ratio is influenced by pH and temperature.

Nitrification. The biological conversion of ammonium to nitrate nitrogen is called Nitrification. Nitrification is a two-step process. Bacteria known as Nitrosomonas convert ammonia and ammonium to nitrite. {2NH3 +3 O2 → 2HNO2 +2 H2O +158 kcal (660kJ). }

Next, bacteria called Nitrobacter finish the conversion of nitrite to nitrate. The reactions are generally coupled and proceed rapidly to the nitrate form; therefore, nitrite levels at any given time are usually low.        {HNO2 + 1/2 O2 = HNO3 – ⊿ G = 18 kcal. }
As we can see the above chemical equation, These bacteria known as “nitrifiers” are strict “aerobes,” meaning they must have free dissolved oxygen to perform their work. Nitrification occurs only under aerobic conditions at dissolved oxygen levels of 1.0 mg/L or more.

These two types of bacteria obtain the energy from the above oxidation process, but the energy utilization is not high, resulting in slow reproduction time. (i.e  more than 10 hours)

So to say, nitrification can only be completed by the combined effect of these two types of bacteria. We knew that the ammonia is harmful to human and fish, How about nitrite? Nitrite poisoning inhibits the uptake of oxygen by red blood cells – known as brown blood, eventually cause severe dealth in fresh water fish. Besides, nitrite can react with metal ions forming salt, and this salt tends to combine with amines, eventually forming a substance with strong carcinogenic effect- alkylene nitramine. Understanding the lethal effect of ammonia and nitrite residue, we have to manipulate the effectiveness of those beneficial bacteria, which in turn increases the efficiency of the nitrogen cycle.
Role of nitrifying bacteria in Aquaculture?

Nitrifying bacteria highly demand oxygen to survive, they love to stay in a variety of fiber cotton, glass rings (silicone made), ceramic microporous filter, where they will get a higher concentration of oxygen exchange. Here is a very important key note for those want to begin their aquaculture farming. The presence of toxic substances in the breeding pool, mainly ammonia and nitrous acid (nitrite), but thank God that this two toxic substances consumed by nitrifying bacteria, and generates a non-toxic nitrate, nitric acid where these substances can directly absorbed by water algae and plankton.

From the ecological point of view of the water, we cannot prevent the ammonia generated.

The more the loading of organic food, the more ammonia generated!!

But you can try to increase the number of nitrifying bacteria to consume the increasing ammonia in the water. Many people disregard the importance the issue, thinking that the more input of the food, the more fast growing of the fish, eventually resulting in losing tones of money.

 

So, what should we do to improve the number of nitrifying bacteria?

The first thing is we must get nitrifying bacteria a fundamental living bed.

Nitrifying bacteria tends to be attached to porous surface fixture, if placed in the pool water for its attachment, it can be quickly attached to the fixture surface and begin to proliferate. They are largely non-motile and must colonize a surface (gravel, sand, synthetic biomedia, etc.) for optimum growth. They secrete a sticky slime matrix which they use to attach themselves.
However, the placement of the fixtures in the pool water is usually not feasible, the reason is that this way may hinder the activities of the fish and is not conducive to fishing pond. More feasible approach is to place a bio ball in a external filtration system, act as breeding bed for nitrifying bacteria.

Denitrification. The biological reduction of nitrate (NO3) to nitrogen gas (N2) by facultative heterotrophic bacteria is called Denitrification. “Heterotrophic” bacteria need a carbon source as food to live. “Facultative” bacteria can get their oxygen by taking dissolved oxygen out of the water or by taking it off of nitrate molecules.