considered a possible vector for the introduction of pathogens into the rearing systems. Artemia nauplii carry a large bacterial load that may be transferred from live preys into
the tanks of fish and shellfish larvae. Some bacteria have been reported to be the source of diseases and high mortalities in fish larvae, and live feeds are thought to be
Ž .
responsible Tatani et al., 1985; Muroga et al., 1987; Nicolas et al., 1989 . Therefore, Artemia nauplii are often treated in order to reduce the bacteria associated with them
prior to feeding them to the larvae. Previous studies often recommend rinsing the nauplii Ž
. in sterile fresh or seawater Austin and Allen, 1982; Rodriguez et al., 1991 , but some
Ž authors argue that rinsing has little effect on the bacteria Dehasque et al., 1991;
. Verdonck et al., 1991 . In this situation, antibiotics have been tried to disinfect the live
Ž feed before introducing into the rearing systems Hatai et al., 1981; Yamanoi and
. Sugiyama, 1987; Tanasomwang and Muroga, 1989, 1992; Gomezgil et al., 1994 . The
potential consequences of antibiotic use in the treatments are the development of antibiotic resistant micro-organisms, multiple antibiotic resistance, resistance transfer to
pathogenic bacteria, and reduced efficacy of antibiotic treatment for diseases caused by
Ž .
resistant pathogens Frappaolo and Guest, 1986 . The research on the development of antibiotic resistant bacteria has mainly focused
on cattle, poultry and swine. Studies on the development of antibiotic resistance in bacterial fish pathogens has also been reported from all areas of aquaculture, ranging
Ž from warm water to coldwater, marine to freshwater Rahim et al., 1984; Hjeltnes et al.,
1987; Tsoumas et al., 1989; McPhearson et al., 1991; Richards et al., 1991; Spanggaard .
et al., 1993 . The purpose of the present study is to determine the antibiotic resistance in bacteria isolated from Artemia nauplii, to find out the alternative for the antibiotics used
routinely in aquaculture systems, to compare the efficacy of formaldehyde to control the bacteria associated with Artemia nauplii and to determine the tolerance level of
Artemia nauplii to formaldehyde and its optimum exposure time to reduce the bacteria.
2. Materials and methods
2.1. Hatching of Artemia cyst Ž
. Artemia cysts Artemia franciscana were obtained from Argent Chemical Laborato-
Ž .
ries, USA. The cysts were hatched in filtered artificial seawater 30 ppt salinity at 288C–308C. After 24 h incubation, instar I nauplii were hatched out from the cysts.
2.2. Bacteriological analysis Groups of 100 freshly hatched nauplii were counted aseptically, placed in a sterile
tissue homogenizer with 1 ml of sterile seawater and homogenized. After homogeniza- tion, the samples were serially diluted to 10
y5
for the estimation of total aerobic w
heterotrophic bacterial flora. In the present study, seawater nutrient agar Bacto-peptone Ž
. Ž
. Ž
. Difco , 5.0 g; yeast extract Difco , 2.5 g; ferric phosphate, 0.1 g; Bacto-agar Difco 15
x Ž .
g, and seawater 1000 ml Oppenheimer and Zobell, 1952 and Thiosulphate Citrate Bile Ž
. Salt agar TCBS-Hi-Media, India prepared in seawater were used for bacterial growth.
Ž Natural seawater was used for media preparation. It was pumped from the sea Bay of
. Bengal near Chennai and allowed to sediment to get rid of sand and other particles. In
the laboratory, the seawater was filtered through filter paper and used. Salinity varied between 30 and 34 ppt, and for media preparation, the salinity was adjusted to 30 ppt
with fresh water. The pour plate technique was followed for estimating the total aerobic heterotrophic bacteria on seawater nutrient agar and the spread plate method was
followed for growth of Vibrio spp. on TCBS agar. After incubation for 48–72 h at
Ž .
298C, plates with 30 to 300 colony forming units CFU were counted. Three
replicates of each dilution were made. After counting, randomly selected colonies Ž
. 10–20rplate were inoculated onto seawater nutrient agar plates. Purified cultures were
maintained on seawater nutrient agar slants at 48C for further studies. 2.3. Antibiotic resistance determination
Ž .
Mueller Hinton agar Hi-Media, India prepared in seawater was used to determine the antibiotic resistance in bacteria isolated from Artemia nauplii. Antibiotics were
mixed with molten agar at approximately 458C or were first diluted in sterile distilled Ž
. water and then added to the molten agar. Antibiotics and dosage levels mgrml
Ž . Ž
. employed in this investigation were chloramphenicol 30
Parke Davis , erythromycin Ž
. Ž .
Ž . Ž
. Ž
. Ž .
15 IPCA , nitrofurazone 100
Hi-Media , oxytetracycline 30 Pfizer and tetra-
Ž . Ž
. cycline hydrochloride 30
Sarabhai Chemicals . The antibiotic plates and control plates without antibiotics were inoculated in duplicate consecutively. The plates were incu-
bated at 298C for approximately 24 h, and the drug resistance was determined. An organism was considered resistant to an antibiotic only if it grew as well on the
antibiotic plate as on the control plate. After antibiotic resistance, twenty bacterial isolates from each resistance profile were selected and identified according to the
Ž .
Ž .
taxonomic schemes of Oliver 1982 and Buchanan and Gibbons 1984 . Ž
. The Minimum Inhibitory Concentration MIC of antimicrobials against the bacteria
isolated from Artemia nauplii were determined by the tube dilution method, in seawater peptone broth composed of 1 peptone and 0.05 beef extract. Varying concentrations
Ž .
0.125, 0.25, 0.5, 1.0, 5.0, 10.0, 20.0, 50.0, 100.0, 150.0, 200.0 and 250 mgrml of chloramphenicol, erythromycin, nitrofurazone, oxytetracycline, tetracycline, formal-
dehyde and sodium hypochlorite were used to determine the MIC of these antimicrobials against bacterial isolates. Two separate dilution series in each antimicrobial agent were
employed and average value of MIC was determined.
2.4. Effect of antimicrobials on Artemia nauplii The toxicity of chloramphenicol, erythromycin, oxytetracycline, tetracycline, nitrofu-
razone, formaldehyde and sodium hypochlorite was tested by bathing the Artemia larvae in antimicrobial treated seawater for 24 h. Batches of healthy nauplii at a density of
Ž .
100r400 ml were reared in beakers 500 ml containing seawater. The physico-chemical characteristics of the rearing medium were salinity, 30 ppt; pH, 8.0–8.1; temperature,
288C–308C and dissolved oxygen, 4.7–5.6 mlrl. After 1 h of acclimatization, pre- weighed antimicrobials were dissolved in the water. Three replicates for each concentra-
Table 1 Ž .
Percentage of bacteria resistant to individual antibiotics. The number of isolates is shown in parenthesis Antibiotic and dosage
Percentage of Ž
. mgrml
resistant bacteria Ž
. Ž
. Chloramphenicol 30
42.9 144 Ž
. Ž
. Erythromycin 15
60.2 202 Ž
. Ž
. Nitrofurazone 100
69.2 233 Ž
. Ž
. Oxytetracycline 30
71.5 240 Ž
. Ž
. Tetracycline 30
21.4 72
tion, including the control, were carried out. The nauplii were fed with rice bran and yeast. The percentage survival of larvae was determined by counting the live larvae after
24 h of treatment and microscopic examination was also made to observe the condition of larvae.
2.5. Antimicrobial efficacy tests The efficacy of formaldehyde to control the bacteria associated with Artemia nauplii
was tested and compared with that of other antimicrobials. Artemia nauplii were collected immediately after hatching and gently washed in sterile seawater. Groups of
Ž .
nauplii 1000rgroup were transferred to the different antimicrobial solutions prepared in seawater. The concentration of antimicrobials used in this test was determined based
Ž .
on MIC and LC50 values Tables 2 and 5 . The treatment was carried out for 8 h and Ž
. larval samples 100 naupliirsample were collected at 0,2,4,6 and 8 h after exposure to
estimate the bacterial flora associated with nauplii. The estimation of bacteria was done as mentioned earlier. Controls consisting of nauplii treated in seawater only were run in
parallel during all experiments.
Table 2 Resistance profiles of 336 strains isolated from Artemia nauplii to various antibiotics and minimum inhibiting
concentrations of formaldehyde and sodium hypochlorite on bacteria isolated from Artemia nauplii. ND — not done
Antibioticsr No. of
Percentage of strains growing at different concentrations of antimicrobials Ž
. antimicrobials
isolates mgrml
0.125 0.25 0.5 1.0
5.0 10.0 20.0 50.0 100.0 150.0 200.0 250.0
Chloramphenicol 336 100
100 100
100 91.7 66.7 50
25 18.4
12.5 Erythromycin
336 100
100 100
100 100
94.6 59.6 42.3 26.8 16.7
8.3 4.8
Nitrofurazone 336
100 100
100 100
81.5 73.9 69.6 65.3 61.8 59.6
56.8 53.6
Oxytetracycline 336
100 100
100 100
100 95.2 80.4 60.1 47.0
33.3 4.8
Tetracycline 336
100 100
95.2 88.1
73.2 68.4 42.9 11.9 8.3 Formaldehyde
336 100
100 94.3
88.2 78.3 66.1 38.7 0
Sodium 336
100 100
82.3 72.7
58.9 55.4 47.0 ND ND
ND ND
ND hypochlorite
Table 3 Percentage composition of bacteria isolated from Artemia nauplii and no. of isolates of prevalent bacterial
genera resistant to various antibiotics. Ch, Chloramphenicol; Er, Erythromycin; Nf, Nitrofurazone; Ot, Oxytetracycline, Tc, Tetracycline
Organisms Composition,
No. of isolates resistant to Ch
Er Nf
Ot Tc
Aeromonas 11
4 7
8 8
3 Alcaligenes
16 5
9 10
8 4
Bacillus 3
2 1
3 1
1 Chromobacterium
4 3
2 2
2 1
Cytophaga 3
1 2
2 1
1 FlaÕobacterium
3 2
2 2
2 1
Moraxella 2
1 2
2 1
1 Pseudomonas
10 5
6 8
5 3
Vibrio 36
17 22
28 24
15 Unidentified
12 –
– –
– –
2.6. Statistical analysis The statistical significance of the difference in efficacy between formaldehyde treated
group and other antimicrobial treated groups was determined with the help of the Student’s t-test. The LC50 values of various antimicrobials for Artemia nauplii were
Ž .
calculated with a computer programme Trevors and Lusty, 1985 based on the method Ž
. described by Finney 1952 .
3. Results
