EFFECTS OF ANTIMICROBIAL AGENTS ON THE ACTIVITY AND SURVIVAL OF AEROMONAS HYDROPHILA AND NITRIFYING BACTERIA

The effects of four common antimicrobial agents (chloramphenicol, gentamycin sulphate,.methylene blue, neomycin sulphate) on the activity and survival of Nitrosontonas, Nitrobacter and Aeron~onas hydrophila isolates from a tilapia fish pond were evaluated. This was done by monitoringgrowth on agar plates and changes in pH, ammonia, nitrite, nitrate and dissolved oxygen concentrations of culture water in experimental flasks during treatment. Ammonia and nitrite oxidations were inhibited by 13.3 mg/l of chloramphenicol, 66.7 mgll of neomycin sulphate and 8 mgll of methylene blue. Gentamycin sulphate (5.3 mgll) had no effect on nitrification. With the exception of gentamycin sulphate, all the other antimicrobials inhibited the growth of Nitrosontonns, Nitrobacter and Aeronzortas hydrophila during treatment.


INTRODUCTION
Species of genera Nitrosomonas, Nitrobacter and Aeromonas are considered autochthonous inhabitants of aquatic environments.They have been isolated in both polluted and non-polluted waters.'These nitrifying bacteria are of critical importance in nitrogen cycling in nature.The concentration as well as form of nitrogenous compounds are among the factors influencing the productivity of an aquatic environment.Microbial populations in tilapia culture pond have been reported to have wide substrate specifi~ity.~Nitrification is carried out exclusively by microbes, the most important of which are the chemolitotrophic nitrifying bacteria typified by the ammonia-oxidizing genus Nitrosontonas and nitrite oxidizing genus Nitrobacter.These organisms are considered to be the most important nitrifiers but their appearance may simply represent successful competition with other nitrifiers such as Aeromonas hydroplzila which can reduce nitrate aerobically by assimilatory reduction via nitrite to ammonium i~ns.~Aeromonashydroplzila has been implicated in several diseases of fish such as haemorrhagic septicaemia, haemolytic bleeding,4 brown patch disease of the skin5 and fin rot.6 The role ofAeromonas hydrophila inmortalities of stressed fish has also been reported."'Antibacterial agents that have found wide usage in fish culture are chloramphenicol, erythromycin, potassium permanganate, gentamycin sulphate, neomycin sulphate, nifurpirinol and methylene b l ~e .~-l l Administration of most of these agents in feed or culture water system is the most practical method of treating systemic bacterial diseases of fish.Hence the question arises as to the effect of these agents on nitrifying bacteria in a fish pond.It has been found that certain antibacterial compounds can inhibit or arrest oxidation of ammonia and nitrite in closed fresh water culture system^'^.'^and in culture flasks inoculated with nitrifying bacteria.llThe inhibition of nitrification in closed culture systems leads to the accumulation of ammonia andlor nitrite substances that can be highly toxic to aquatic animals.I3 No published work so far has associated Nitrosomonas and Nitrobacter with any form of disease in both plants and animals.However, when bacteriological filtration is interrupted by chemotherapy, the resulting deterioration in water quality may be more lethal than the pathogens to the diseased animals.14This study was undertaken to determine the effect of four commonly used antimicrobial agents on the activity and survival of Nitrosomonas, Nitrobacter and Aeromonas kydrophila isolated from tilapia fish pond in order to assess their effect on the cycling of nitrogen in the environment.

METHODS AND MATERIALS
Sample Collection: Studies were conducted with pond water samples collected at Duru Fish Farms located on the bank of Njaba River at Awo-Omamma, Oru Local Government Area of Imo State, Nigeria where approximately 6000 young active tilapia were reared in fresh water.Also disinfection ofthe pond water with antimicrobial agents is carr$ed out twice in a year.Pond water samples were analysed for the following pfiysico-chemical parameters: pH, ammonia, nitrite, nitrate and dissolved oxygen (DO) concentrations.Measurements were made using Direct Reading Engineering Laboratory(DR-ELIS) Hach kit.'"Culture Studies: Serial dilutions of the samples were aseptically carried out at the University of Port Harcourt using sterile physiological saline (0.85% wlv NaC1) as diluent before plating on appropriate media.Each flask contained the three bacterial species.Nitrifying bacterial counts were estimated on agar plates treated with mineral salt medium containing either ammonium sulphate or sodium nitrite for Nitrosomonas and nitrite oxidizing Nitrobacter respectively.16 Presumptive Aeromonas hydroplzila was enumerated on RS agar17 and medium of Kaper et al. (1979).Plates were incubated following the spread plate technique.Inoculated plates were incubated a t 30°C for 4 days.Reported plate counts were those lying within the range 30-300 colony forming units (cfu).Colonies were randomly selected from each medium, isolated and restreaked on fresh selective media plates to purify.Pure cultures were then maintained at 4OC as stock cultures for further tests on the selective media.Characterization ofthe isolates was performedlg and involved cell micromorphology, Gram reaction, motility, oxidase, catalase, urease, H,S production, citrate utilization, indole, methyl red-Voges Proskauer(MR-VP) tests, oxidationlfermentation of glucose and decarboxylase tests.
Survival Studies: Survival studies of pure cultures of the three bacterial isolates on the antimicrobials (shown below) were carried out in culture flasks.

Anti7nicrobial
Dosage (mgA) Neomycin sulphate Chloramphenicol Methylene blue Gentamycin sulphate The experimental methods similar to those d e ~c r i b e d l ~. ~~ were employed.Nine 1-litre Erlenmeyer flasks were each filled with 500 ml of pond.water, sterilized by filtration through a 0.22 pm pore size membrane (Millipore Corp) and autoclaved a t 121°C for 30 minutes.They comprised four sets of two flasks per set and one control flask.Pure culture suspensions in sterile water were made separately in sterile 50 ml flasks with each ofthe bacterial species isolated from the tilapia pond water.The bacterial suspensions were aseptically serially diluted to and the counts of this dilution determined by plating onto agar plates containing selective media and then adjusting to 10" cells ml-'.One millilitre of the bacterial suspension with cell concentration of 10"l-' was inoculated into 500ml of each of a set of two flasks.No organisms were added to the control flask.
The antimicrobial agents a t concen.trationsfrequently used for the treatment of diseases of fresh water aquarium fishesS were added directly to the sterilized pond water in culture flasks as single dosages on day zero.Each of the four antimicrobials was added to one set of two flasks and no antimicrobials to the control set.The concentrations of NH; -N , NO, -N, NO, -Nand pH were all determined immediately before the bacterial suspension and antimicrobials were added on day zero and on alternate days thereafter for the duration of the experiment which lasted 21 days.
All the flasks were capped with cotton wool bungs and aerated by shaking a t 120 rpm.On alternate days, 0.1 ml of water from each flask was plated on selective medium for survival studies.Colonies per plate were counted after incubating a t 30°C for 48 h.Bacterial activity was monitored by determining changes in the pH and concentrations of NH' -N, NO, -N, NO, -N.Approximately 150 ml of water removed for samplkg were replaced once weekly with newly mixed sterilized pondwater containing appropriate concentrations of the antimicrobials.
Statistical analysis: Mean bacterial counts were subjected to log,, transformation and stepwise multiple linear regressions were calculated using the IBM Computer (Model-30).The mean values thus obtained in the analysis were compared using F-test to evaluate the statistical significance.

RESULTS
The effects of the various antimicrobials on the chemical parameters measured over an 18-day period following treatment in experimental flasks are given in Fig. 1.
The filter-sterilized pond water quality in all the flasks prior to treatment was: In control systems no significant changes in water quality were observed since they were not inoculated with bacteria.The pH gradually declined from 6.73 on day zero to 6.70 on day 18.The pH values in flasks treated with antimicrobial agents did not differ significantly (p>0.05) from those of the controls.
Treatment offlasks with gentamycin sulphate (5.3 mgA) did not significant1.yalter water quality.Flasks treated with neomycin sulphate (66.7 mgA) resulted in water quality changes significantly different from the control (p < 0.05).Total ammonia-nitrogen rose to a peak of 1.38 mgAon day 8 and thenrapidlydecreased (Fig. 1).Nitrite-nitrogen rose immediately during treatment to a peak of 2.5 mg/l on day 8 and rapidly decreased to 0.15 mgll on day 18.Nitrate-nitrogen fell immediately after treatment to 6.0 mg/l on day 2, remained relatively stable until day 10 when it began rising rapidly reaching 13.0 mgA on day 18.
Treatment with methylene blue (8.0 mgd) resulted in water quality changes similar to those oftlle neomycin sulphate.Total ammonianitrogen rose to apeak of 0.88 mgtl on day 6 and then gradually declined.Nitrite-nitrogen remained stable.Nitrate-nitrogen rose during treatment reaching 13.5 mg/l N O -N on day 18 (Fig. 1).Treatment with chloramphenicol(13.3mgA) resulted in higher concentrations of total-ammonia nitrogen.The nitrite-nitrogen values did not differ significantly (p>0.05) from those of controls.Nitrate-nitrogen rose immediately during treatment reaching 14.2 mgA on day 18.

T i m e (days)
Survival studies for pure cultures of the three bacterial isolates were conducted with the antimicrobial agents.The data for survival of the three bacteria as measured indirectly on agar plates are plotted in Fig. 2.
Gentamycin sulphate did not inhibit the growth ofNitrosomonas.However, it had some inibitory effect on Nitrobacter and more on Aeromonas Izydrophila.Chloramphenicol caused marked inhibition of the growth of all three organisms.With neomycin sulphate, the numbers ofNitrobacter andAritrosomonas declined rapidly during treatment.This agent had a strong effect on Aeromonas hydrophila.Methylene blue caused marked inhibition of the growth of Nitrosomonas, Nitrobacter and Aerornonas hydrophila.
A significant (p <0.05) negative correlation was found to occur with N H ~ -N and NO, -N; these parameters demonstrated the greatest influence with respect to the survival of the three bacteria in flasks treated with antimicrobial agents.

DISCUSSION
Some of the antimicrobial agents decreased in the concentrations of ammonia, nitrite and nitrate.The pH values in the treated culture flasks did not differ significantly from those of the control.Gentamycin sulphate did not inhibit the growth ofNitrosomolzas but slightly inhibited Nitrobacter and more so A. Izydroplzila.These results corroborate the observation of Bower & Turner (1981) that gentamycin sulphate has no significant effect on nitrification in a recirculating aquatic system.
Chloramphenicol at a concentration of 13.3 mgA inhibited all three organisms.The results are consistent with those of Levine Meade (1976) who reported 48% inhibition in mixed culture of nitrifiers treated with 12.5 and 15.0 mgA chloramphenicol.They also agreed with the report of Collins et al. (1976) who found no inhibition of ammonia oxidation in fresh water aquaria by 50 mgA chloramphenicol.
Neomycin sulphate at 66.7 mgA inhibitednitrite oxidation and the numbers of Nitrobacter declined rapidly.This antimicrobial agent s1ightl.yinhibited ammonia oxidation and the numbers of Nitrosomonas.However, it has severe effects onAeromonas hydrophila.This suggests that neomycin sulphate is toxic to the three isolates.These results corroborate the observation of Bower & Turner (1982) that similar treatment dosage inhibited nitrite oxidation and slightly inhibited ammonia oxidation in fresh water aquaria.
In this study, 8 mgll methylene blue caused marked inhibition of the growth of Nitrosomolzas, Nitrobacter and A. lzydrophila and thus ammonia and nitrite oxidation in fresh water aquaria.These results are in consonance with those of Levine & Meade (1976) that 2.0 mgll methylene blueinhibits nitrification by 92% in culture, and they also agree with the observation of Collins et al. (1976) that 5 mgA methylene blue inhibits ammonia oxidation in freshwater aquaria.
With the exception of gentamycin sulphate, nitrogen transformation was markedly inhibited in all the experimental flasks treated with other antimicrobial agents.Increase in ammonia with stable nitrate levels indicated that nitrogen transformation had ceased.Elevated levels of nitrite lasted for about 14 days in all the flasks until the nitrite was probably converted to nitrate by the recovery of few surviving Nitrobacter which oxidized nitrite to nitrate.The pattern of water quality changes is similar to that described by Collins et al. (1976) in an unconditioned aquatic recirculating system indicating that both Nitrosomonas and Nitrobacter as well as other bacteria in the system were inhibited or killed by the antimicrobial agents.
The results of the experiment clearly demonstrate that the majority of the generally accepted antimicrobials for disease treatment in aquaculture systems can have adverse effects on nitrogen transformationin those systems since they are highly toxic to Nitrosolnolzas and Nitrobacter.Their inhibition leads to the accumulation of ammonia and or nitrite, substances that can be highly toxic to aquatic animals.The use of methylene blue, neomycin sulphate, or chloramphenicol is, therefore, not recommended for the control of diseases in such culture ponds.The results show clearly that gentamycin sulphate is the preferred agent for the treatment of A. lzydrophila infections in fish culture ponds in Nigeria since it has minimal effect on nitrogen cyclingin the fresh-water ecosystem.