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
Total numbers of the aerobic heterotrophic bacterial flora of Artemia nauplii were estimated on seawater nutrient agar and TCBS agar. The total number of bacteria ranged
Table 4 Percentage mortality
a
of Artemia nauplii exposed for 24 h by bath treatment against antimicrobials at Ž
. different concentrations, and 24 h LC50 values mgrl . ND — not done
Percentage mortality of Artemia nauplii Ž
. Antimicrobials
Concentrations mgrl used 24 h LC50 values
Ž .
mgrl with 95 2
4 8
16 50
100 200
400 800
confidence limit Chloramphenicol
ND ND
ND ND
ND 5.7
14.7 21.3
47.4 Not determined
Ž .
Oxytetracycline 0.3
ND ND
ND ND
ND 0.3
6.7 13.0
84.7 540.5 503.7, 579.9
Tetracycline ND
ND ND
ND ND
1.7 10.0
19.0 26.3
Not determined Erythromycin
ND ND
ND ND
ND 17.7
32.7 38.0
46.7 Not determined
Nitrofurazone 1.3
ND ND
ND ND
ND 28.3
36.7 42.0
45.7 Not determined
Ž .
Formaldehyde ND
ND ND
ND 27.3
31.3 41.3
57.7 ND
293.1 226.0, 380.0 Ž
. Sodium hypochlorite
1 2.3
18 28.7
100 ND
ND ND
ND ND
5.6 5.4, 5.9
a
Mean of three replicates.
A.S. Sahul
Hameed, G.
Balasubramanian
r Aquaculture
183 2000
195 –
205 200
Table 5 Ž
.
U
Estimated number of bacterial cells thousands of CFU per Artemia nauplius associated with Artemia nauplii in the absence and presence of antimicrobials at
different time intervals. Values are meanS.E. SNA — seawater nutrient agar, TCBS — TCBS agar Ž .
Time h Culture media used
Name and concentration of antimicrobial used
a a
a a
a
Without Chloramphenicol
Erythromycin Oxytetracycline
Tetracycline Formaldehyde
Ž .
Ž .
Ž .
Ž .
Ž .
antimicrobials 150 mgrl
200 mgrl 200 mgrl
100 mgrl 50 mgrl
SNA 4.80.23
4.80.23 4.80.23
4.80.23 4.80.23
4.80.23 TCBS
2.30.21 2.30.21
2.30.21 2.30.21
2.30.21 2.30.21
2 SNA
5.60.23 0.130.006
0.50.02 0.340.04
0.170.02 0.730.08
2 TCBS
2.60.26 0.070.003
0.080.003 0.050.002
0.010.002 0.50.003
4 SNA
6.20.23 0.110.01
0.330.03 0.10.04
0.0030.0003 0.50.02
4 TCBS
3.00.34 0.070.002
0.030.004 0.30.01
6 SNA
6.80.33 0.080.005
0.070.002 0.060.003
0.0050.0003 0.050.001
6 TCBS
3.60.38 0.0010.00008
0.010.0008 8
SNA 6.70.34
0.030.003 0.040.002
0.020.003 0.020.003
0.030.003 8
TCBS 3.90.41
0.0040.0002
U
Mean of three replicates.
a
Significantly different from control, p- 0.001.
from 3.8 = 10
3
to 8.1 = 10
3
CFU per nauplius on seawater nutrient agar and 9.4 = 10
2
to 4.3 = 10
3
CFUrnauplius on TCBS agar. A total of 336 isolates was examined for their resistance to five antibiotics. The frequencies of resistances to individual antibiotics
are shown in Table 1. More than 60 of the bacterial isolates were resistant to erythromycin, nitrofurazone and oxytetracycline.
The MIC values of seven antimicrobials against the bacterial isolates were deter- mined and the results are presented in Table 2. The growth of all bacterial isolates was
inhibited by chloramphenicol at 200 mgrl, oxytetracycline at 250 mgrl, tetracycline 150 mgrl or formaldehyde at 50 mgrl. Erythromycin inhibited the growth of 95.2 of
bacterial isolates at 250 mgrl; nitrofurazone at 250 mgrl and sodium hypochlorite at 20 mgrl inhibited the growth of 46.4 and 53 of the bacterial isolates, respectively.
The bacterial isolates were identified to at least generic level and the results are given in Table 3. The percentage of isolates of each genus resistant to each antibiotic is shown
in Table 3. Percentage mortality of Artemia nauplii exposed for 24 h at different concentrations
of antimicrobial agents is given in Table 4. High mortality of Artemia nauplii was Ž
. observed when the larvae were exposed to chloramphenicol 800 mgrl , oxytetracycline
Ž .
Ž .
Ž .
Ž 800 mgrl , erythromycin 800 mgrl , nitrofurazone 800 mgrl , formaldehyde 400
. Ž
. mgrl or sodium hypochlorite 16 mgrl . Chloramphenicol, oxytetracycline, tetra-
cycline, formaldehyde and sodium hypochlorite caused significant mortality in higher concentrations. Nauplii with broken setae and appendage deformities were encountered
when the larvae were exposed to higher concentrations of antimicrobials. The LC50 values of various antimicrobials for Artemia nauplii were determined and the results are
presented in Table 4. The LC50 values of oxytetracycline, formaldehyde and sodium hypochlorite recorded were 540.5, 293.1 and 5.6 mgrl, respectively, after 24 h of
exposure.
The effect of antimicrobial agents on the bacteria associated with the Artemia nauplii was tested and the results are presented in Table 5. The antimicrobials significantly
reduced the bacterial load of Artemia on seawater nutrient agar and TCBS agar when compared to the control. In the absence of antimicrobials, the bacterial flora of Artemia
nauplii increased to level of 6.7 = 10
3
and 3.9 = 10
3
rnauplius on seawater nutrient agar and TCBS agar, respectively, after 8 h of incubation. There is no significant differences
between antibiotic and formaldehyde treatments to disinfect the Artemia nauplii.
4. Discussion
