Aquaculture 195 Ž2001.

193–204
www.elsevier.nlrlocateraqua-online

Antibiotic resistance of bacteria from shrimp ponds

˜
Eleonor A. Tendencia) , Leobert D. de la Pena
Aquaculture Department, Southeast Asian Fisheries DeÕelopment Center, Tigbauan, Iloilo, Philippines
Received 1 March 2000; received in revised form 18 October 2000; accepted 7 November 2000

Abstract

The incidence of antibiotic resistance was compared in bacteria isolated from pond water, pond
sediment, water and sediment from the receiving environment Žarea where water from pond
drains, which is 0 and 50 m away from the exit gate, in this study. and cultured shrimp from
ponds that have not used any antimicrobials, ponds that have previously used antimicrobials and
ponds that are currently using oxolinic acid. Most of the bacteria isolated from all sample and
pond type were Vibrios. Among the Vibrios, V. harÕeyi were most commonly isolated. Multiple
antibiotic resistance ŽMAR. to at least two antimicrobials was highest in ponds currently using
oxolinic acid Ž24% of bacteria isolated from such ponds., followed by those that have previously
used antimicrobials Ž19%. and the least was those from ponds that have not used any antimicro-
bials Ž17%.. The lowest incidence of antibiotic resistance was observed in ponds that have not
used any antimicrobials Ž41% of the isolates from such ponds.. Among the individual antibiotics,
incidence of resistance to oxytetracycline was highest Ž4.3% of the total number of isolates.
followed by furazolidone Ž1.6%., oxolinic acid Ž1%. and chloramphenicol Ž0.66%..
Resistance to individual chemotherapeutants did not reflect the pattern of antimicrobial use
with ponds that have previously used antimicrobials showing the highest incidence of resistance to
one antimicrobial Ž12% of total isolates from such ponds.. Resistance to both oxolinic acid and
furazolidone Ž15% of total number of isolates. was highest compared to other antimicrobial
resistance profiles Ž1–12%.. Multiple antimicrobial resistance and intermediate reaction to at least
one antimicrobial are associated with antimicrobial use. q 2001 Elsevier Science B.V. All rights
reserved.

Keywords: Antibiotic resistance; Shrimp

)
Corresponding author. Southeast Asian Fisheries Development Center, P.O. Box 256, 5000 Iloilo City,
Philippines.
˜ ..
E-mail addresses: sidgigit@iloilo.net ŽE.A. Tendencia., leobertd@aqd.seafdec.org.ph ŽL.D. de la Pena

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 0 0 . 0 0 5 7 0 - 6
194 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena

1. Introduction

Shrimp farming in the Philippines has been a major success in the 1980s until its
decline in the mid-1990s. The growth of the shrimp aquaculture industry increased the
need to intensify farming practices to maximize profits. Problems of diseases often
accompanied this intensification as environmental conditions deteriorated and brought
the decline of the industry. Pressure to ensure production led to reliance on antibiotics.
Chemotherapy is widely practiced in the Philippines ŽBaticados and Paclibare, 1992..
Antibiotics are administered to farmed shrimp primarily to prevent or treat bacterial
diseases. In Philippine grow-out ponds, oxytetracycline, oxolinic acid, chloramphenicol
and furazolidone are incorporated in artificial feeds as treatment against luminescent
vibriosis. Other antibacterial agents used in intensive prawn farms in the Philippines
include the nitrofurans, erythromycin, and sulfa drugs ŽBaticados et al., 1990; Primavera
et al., 1993..
The presence of antimicrobial agents at low concentration through leaching or
continued usage may lead to the development of drug-resistant strains and multiple
antibiotic resistance ŽMAR. in bacteria, which may result to resistance transfer to
pathogenic bacteria and reduced efficacy of antibiotic treatment for human and animal
diseases. Several studies have been done to investigate the possible consequences of the
use of antimicrobials. Most of the studies are focused on fish and its environment.
Spanggaard et al. Ž1993. reported resistance to oxytetracycline among bacteria from
freshwater fish farms in Denmark. Nygaard et al. Ž1992. reported that exposure to
oxytetracycline and oxolinic acid initiates resistance to other drugs. McPhearson et al.
Ž1991. observed that individual and multiple antibiotic resistances were associated with
antimicrobial use. Antibacterial-resistant bacteria in surficial sediments near salmon net
cage farms were isolated by Herwig et al. Ž1997., suggesting that these antimicrobials
may increase the level of resistance in bacteria in the surrounding environments.
According to Kerry et al. Ž1995., resistant flora in feed can, under certain circumstances,
significantly contribute to the resistant flora detected in sediments under fish cages.
Kerry et al. Ž1997., on the other hand, reported that emergence of strains resistant to
oxytetracycline in the microflora of the intestine of Atlantic Salmon held in seawater is
not a necessary consequence of its oral administration.
A few studies on antibiotic resistance in shrimp have been done. Baticados et al.
Ž1990. reported that luminous strains of Vibrio harÕeyi and V. splendidus isolated from
shrimp larvae are resistant to erythromycin, kanamycin, pencicillin G, and streptomycin.
Abraham et al. Ž1997. were able to isolate V. harÕeyi strains from diseased shrimp that
are resistant to ampicillin, chlorotetracycline, ceprofloxacin, erythtromycin, furazoli-
done, gentamycin, nalidixic acid, neomycin, nitrofurantoin, nitrofurazole, novobiocin,
ofloxacin, oxytetracycline, penicillin G, polymyxin B, rifampicin, streptomycin, sulpha-
somodine, sulphamethezole, and sulphafurazole. Leano ˜ et al. Ž1999. reported that Vibrio
spp. and Aeromonas spp. from fish and shrimp were resistant to streptomycin but
sensitive to oxolinic acid. They further reported that Aeromonas spp. were also resistant
to oxytetracycline, and trimethoprimrsulphamethoxazole.
The indiscriminate use of antimicrobials calls for a better understanding of the impact
of these compounds on the microflora of the environment and the cultured shrimp.
 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena 195

This study compares the incidence of antibiotic resistance in bacteria from pond
water, pond sediment, receiving water, receiving sediment and cultured shrimp from
ponds that have not used any antimicrobials, previously used antimicrobials and
currently using antimicrobials.

2. Materials and methods

2.1. Sampling site

Shrimp Ž Penaeus monodon., pond water, pond sediments, receiving soil and receiv-
ing sediments were collected from six shrimp farms in: Antique Ž1., Iloilo Ž1., Aklan
Ž1., Capiz Ž1., and Negros Oriental Ž2., Philippines. Samples were taken from one pond
in each farm. The farms were chosen based on the histories of antimicrobial use. Two
ponds have not used any antimicrobials; two have previously used antimicrobials but
had not added any for 3 years prior to sampling; and two are currently using oxolinic
acid incorporated in feeds at 9 grkg of feed given once a week as a prophylactic
treatment.
Ponds that did not use any antimicrobials will be denoted as A and B, ponds that
have previously used antimicrobials as C and D, and ponds that are currently using
oxolinic acid, as E and F. Ponds A and B are far from any other fish or prawn farms,
while farms C, D, E, and F are surrounded by other prawn and milkfish farms. Pond
area of the six ponds varies from 2000 to 4000 m2 . Except for pond C, which is
culturing tilapia in cages inside the prawn ponds, all are into monoculture of P.
monodon. Farms A and B have been in operation for more than 5 years, while farms C
and D had just resumed after 3 years of non-operation. Farms E and F have been in
operation for more than 10 years but are using oxolinic acid for the first time when
sampling was done. All six farms till, dry and lime the ponds during preparation. No
fertilizer is added but teaseeed is applied. Source of water for ponds A, B, D, E and F
are nearby seas and rivers, and an adjacent brackishwater tilapia pond for pond C Žthe
source of water for the tilapia pond are nearby seas and rivers.. All the ponds change
water as needed at 30–50% of total volume. All ponds used commercial pellets to feed
the prawn. During disease outbreak, ponds A, B, C, and D increase the frequency of
water change. Ponds E and F apply oxolinic acid for disease prevention or prophylactic
treatment.

2.2. Sample collection

Water, soil, and shrimp Ž P. monodon. samples were collected by divers during the
rearing period ŽDOC 81 " 51.. Pond water Ž100 ml. was separately collected from three
different places in a pond Žwhere water enters, in the center and where water drains..
Water samples were collected from the surface using 100-ml sterile bottles. Pond
sediments Ž10 ml. were collected from the upper pond bottom from the three places
where pond water was collected. Soil samples were collected by scraping the sediment
196 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena

surface using sterile bottles and capping the container before bringing the sample to the
surface. Receiving soil and receiving water were also collected as in pond water and
soil, 0 and 50 m away from the exit gate following wind direction. Five shrimp samples,
15 " 10 g in weight, were collected from each pond. The shrimps were dissected alive,
the hepatopancreas and lymphoid organs aseptically taken and processed individually.
Samples from ponds A, B, D, and F were processed for bacterial isolation on site,
1–2 h after the samples were taken. Samples from ponds C and E were processed in the
laboratory within 2 h of sampling. Shrimp samples were transported live in plastic bags
with aeration. All samples were transported from the site to the laboratory inside an
air-conditioned car.
Samples were processed for bacterial isolation and identification and antimicrobial
sensitivity test.

2.3. Bacterial isolation

Bacteria from the water, sediment, and shrimp were isolated using the spread plate
technique. Three media were used: 2216 E Zobell’s medium ŽAaronson, 1970., Pseu-
domonas–Aeromonas Selective Agar ŽGSP; Merck. and Thiosulfate Citrate Bile Sucrose
Agar ŽTCBS; Baltimore Biological Laboratories ŽBBL...

2.3.1. Sediment samples

One gram of sediment sample was homogenized in 1 ml autoclaved Ž1218C for 15
min. seawater. The homogenized sample was then diluted and serial 10-fold dilution up
to 10 4 were prepared in autoclaved seawater. Portions Ž0.1 ml. from each dilution were
plated onto the three media in duplicate. Plated samples were incubated for 24–72 h at
358C. After incubation, representative colonies were chosen from each plate and purified
on Nutrient Agar ŽNA; BBL. plate.

2.3.2. Water sample

One milliliter of water sample was serially diluted in autoclaved seawater, plated, and
processed as in sediment samples.

2.3.3. Shrimp samples

Five shrimps from each pond were processed individually. The hepatopancreas and
lymphoid organs were separately taken aseptically from the shrimp and placed in
individual sterile test tubes and homogenized in 3 ml autoclaved seawater using sterile
glass rod. The samples were serially diluted in autoclaved seawater, plated, and
processed as in sediment samples.

2.4. Bacterial storage

Purified samples were stocked in nutrient broth with 20% glycerol and 1.5% Žwrv.
sodium chloride and stored at y808C until use.
 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena 197

2.5. Bacterial identification

Isolates stocked in y808C were streaked onto NA plates and identified using
conventional biochemical tests described by MacFadden Ž1976. and West and Colwell
Ž1984.. One-day-old cultures on NA were used as inocula. The hanging drop method
was used to test motility. Gram stain reaction and cell morphology were observed
following the method by Hucker, as cited by Hendrickson Ž1985.. Tryptic Soy Agar
ŽTSA. ŽBBL., supplemented with 3% Žwrv. sodium chloride, was used to determine the
swarming characteristics of the bacteria. The isolates were identified based on the
scheme by Alsina and Blanch Ž1994. and Baumann and Schubert Ž1984..

2.6. Antimicrobial sensitiÕity test

Isolates stocked in y808C were streaked onto Nutrient Agar plates and processed for
antimicrobial sensitivity test using the disc diffusion method. Procedures were based on
the standardized disc agar diffusion method of the National Committee for Clinical
Laboratory Standards for antimicrobial susceptibility tests ŽFinegold and Martin, 1982..
Isolates were inoculated on Tryptic Soy Broth ŽBBL. and incubated for 2 h. The
turbidity of the bacterial suspension was then compared with a5 MacFarland’s Barium
Sulfate Standard Solution Žcorresponding to 1.5 = 10 8 cfurml.. If the bacterial suspen-
sion was more turbid, autoclaved seawater was added drop by drop; if less turbid, more
bacteria were added to the tube or the inoculated tube was further incubated. The
standardized bacterial suspension was then inoculated onto Muller Hinton Agar ŽBBL.
using sterile cotton swabs, which were then left to dry for 10 min before antimicrobial
sensitivity discs ŽBBL; BBL discs, 6-mm diameter. were applied. The discs used were
oxytetracycline ŽOTC., 30 mg; oxolinic acid ŽOXA., 2 mg; chloramphenicol ŽC., 30 mg;
and furazolidone ŽFx., 100 mg. The plates were incubated for 18–24 h at 358C. After
incubation, the diameter of the zone of inhibition was measured and compared with the
BBL zone diameter interpretative chart to determine the sensitivity of the isolates to the
antibiotics. The BBL zone interpretative chart was used in the absence of standard
interpretative scheme for environmental isolates or for shrimp pathogens. The procedure
is intended for in vitro susceptibility testing of common rapidly growing and certain
fastidious bacterial pathogens. The procedure was adopted from the National Committee
for Clinical Laboratory Standards. Susceptibility tests of antimicrobials not listed in the
chart, as in furazolidone, were interpreted on the basis of the presence or absence of a
definite zone of inhibition.

3. Results

3.1. Generic composition of microflora

A total of 413 isolates were examined for their resistance to four antibiotics.
Ninety-six of the total isolates came from ponds that have not used any antimicrobials,
101 came from ponds that have previously used, and 216 came from ponds that are
198 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena

currently using OXA. Most of the bacteria isolated from all sample and pond type
belonged to the genus Vibrio except those from the pond soil and shrimps of ponds that
have previously used antimicrobials, wherein most of the bacteria recovered belonged to
the genus Aeromonas. Among the Vibrios, V. harÕeyi were most commonly encoun-
tered from all sample and pond types. Bacteria isolated from pond water were usually
isolated from other sample types as well. Bacterial groups isolated from the same pond
type were similar. Acinetobacter, Achromobacter, Alcaligenes, V. alginolyticus, V.
aestuarinus and V. natriegensis were isolated from ponds currently using OXA but not
from ponds that have previously used and have not used any antimicrobial.

3.2. Antibiotic resistance

The percentage of bacteria from incoming water of ponds that showed sensitivity to
all four antimicrobials was highest in ponds that have not used any antimicrobial Ž53%.,
followed by those that have previously used Ž36%. and the least from ponds that are
currently using OXA Ž33%.. Fifty-nine percent of bacteria from ponds that have not
used any antimicrobials showed sensitivity to all four tested chemotherapeutants, 49%
from ponds that have previously used and 40% from ponds that are currently using
OXA. Higher percentage of bacteria isolated from the pond water and soil showed
sensitivity to the four tested antimicrobials than those from the receiving water and soil
of all three pond types. Percentage of bacteria showing resistance to at least two
antimicrobials was highest in ponds that are currently using OXA Ž24%., followed by
those from ponds that have previously used OXA Ž19%., and the least was those from
ponds that have not used any Ž18%.. The incidence of bacteria showing sensitivity to

Table 1
Antibiotic resistance profiles encountered
Resistance profile Percentage occurrence in bacteriaa
Ponds that did not Ponds that previouslys Ponds that are currently Total
use antimicrobial used antimicrobials using an antimicrobial
OTC, OXA, Fx, C 2 0 4 6
OTC, OXA, Fx 3 1 1 5
OTC, OXA, C 0 0 1 1
OXA, C, Fx 0 1 1 2
OTC, C, Fx 0 2 2 4
OTC, OXA 1 0 1 2
OTC, Fx 1 2 9 12
OTC, C 4 7 1 12
OXA, Fx 5 4 6 15
OXA, C 0 0 1 1
C, Fx 0 2 1 3

OTC s oxytetracycline, 30 mgrml; Fx s furazolidone, 100 mgrml; OXA s oxolinic acid, 2 mgrml; C s
chloramphenicol, 30 mgrml.
a
ŽNumber of isolates showing the same profilertotal number of isolates from such ponds.=100; rounded
off to the nearest whole number.
 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena 199

three antimicrobials but intermediate reaction to one was highest in ponds currently
using OXA Ž30% of bacterial isolates from such ponds., followed by bacteria from
ponds that have previously used antimicrobial Ž20%. and the least were bacteria from
ponds that have not used any Ž18%.. Among the individual antibiotics, incidence of
resistance to oxytetracycline was highest Ž4.3% average among the three ponds.,
followed by furazolidone Ž1.6%., oxolinic acid Ž1%. and chloramphenicol Ž0.66%..
Table 1 shows the different antibiotic resistance profiles encountered. Resistance to
both oxolinic acid and furazolidone was highest Ž15%. compared to other antibiotic
resistance profiles Ž1–12%..

3.3. Intraspecies resistance

The percentage resistance of the dominant species or genus from different pond
sources are shown in Table 2. The incidence of resistance among the Vibrios was

Table 2
Percentage resistance of dominant species from different pond source
Bacterial species Pond No. of % Sensitive % % Resistant to % Int a
source isolates to all MAR OXA OTC FX C
Aeromonas N 13 69 8 0 23 0 0 0
P 21 62 29 5 0 0 0 5
C 21 38 19 0 5 5 5 29
Pseudomonas N 21 29 48 5 5 5 0 10
P 10 50 30 0 0 20 0 0
C 74 55 20 1 0 1 0 22
V. harÕeyi N 35 37 11 0 3 0 0 49
P 25 48 12 0 8 4 0 28
C 19 16 32 0 5 0 0 47
V. mimicus N 1 100 0 0 0 0 0 0
P 2 50 0 0 0 0 0 50
C 20 25 25 0 10 0 0 40
V. splendidus N 4 75 0 0 0 0 0 25
P 10 50 20 0 10 10 0 10
C 2 50 0 0 0 0 0 50
V. Õulnificus N 3 33 0 0 33 0 0 33
P 4 50 0 0 50 0 0 0
C 7 57 14 0 0 0 0 29
Other Vibrios N 13 69 0 0 0 0 0 31
P 23 70 22 0 0 0 0 9
C 59 19 27 0 3 0 0 51
Other gram-negative N 6 83 17 0 0 0 0 0
bacteria P 6 0 50 0 33 0 0 17
C 14 36 43 0 7 0 0 14

Ns Ponds that did not use any antimicrobials; P s Ponds that have previously used antimicrobials; C s Ponds
that are currently using antimicrobials; OXA s oxolinic acid, 2 mg; OTC s oxytetracycline, 30 mg; Fx s
furazolidone, 100 mg; C s chloramphenicol, 30 mg; MAR s multiple antibiotic resistance; resistant to more
than one antimicrobials.
a
Percentage showing sensitivity to three antimicrobials but intermediate reaction to one.
200 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena

Table 3
Antimicrobial resistance profile of different ponds
Pond source
A B C D E F
% MAR 25 16 26 15 23 33
% Sensitive to all 50 50 54 53 36 33
% Resistant to
OTC 7 1 0 3 5 7
OXA 4 0 3 2 1 0
Fx 0 1 5 2 1 1
C 0 0 0 0 2 0
% Intermediate 14 31 13 26 33 27
reaction to 1

Ponds A and Bs ponds that have not used any antimicrobials; Ponds C and Ds ponds that have previously
used antimicrobials; Ponds E and F s ponds that are currently using oxolinic acid; OTC s oxytetracycline, 30
mgrml; OXA s oxolinic acid, 2 mgrml; Fx s furazolidone, 100 mgrml; C s chloramphenicol, 30 mgrml;
MAR s isolates showing resistance to two or more antimicrobial.

associated with antibiotic use. For each Vibrio species there was generally a consistent
increase in the percentage of resistant bacteria from ponds that have not used any
antimicrobial to those from ponds that have previously used or are currently using.

3.4. Comparison of bacterial resistance between the two ponds

The antimicrobial reactions of bacteria from the six ponds are presented in Table 3.
The percentage of bacteria showing sensitivity to the four tested antimicrobials did not
vary between the two ponds in each group. However, resistance to different antimicro-
bials varies between the two ponds belonging to the same groups.

4. Discussion

There is no marked difference in the composition of the microflora of the different

pond source except that Acinetobacter, Achromobacter and Alcaligenes were isolated
only from ponds currently using OXA.
Results of the present study indicate that multiple antibiotic resistance and intermedi-
ate sensitivity test reaction to one chemotherapeutant are associated with antimicrobial
use. Results also point out that when antimicrobial agents are used more often in an
environment, the higher will be the occurrence of resistant microorganisms in that site.
This is because exposure to antimicrobials may also result to cross-resistance between
other aquaculture antibacterials. Nygaard et al. Ž1992. reported that exposure to oxyte-
tracycline or oxolinic acid initiates resistance to other drugs. According to Hansen et al.
Ž1992., bacteria exposed to oxolinic acid developed cross-resistance towards flumequine
and oxytetracycline. In 1991, McPhearson et al. reported that the incidence of resistance
was associated with antibiotic use. The frequency and rates of ascent and dissemination
 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena 201

of antibiotic resistance in bacterial population are anticipated to be directly related to the

volume of antibiotic use ŽAnderson and Levin, 1999..
Bacteria from the incoming water of ponds currently using OXA showed the highest
incidence of resistance to antimicrobials, followed by those from ponds that have
previously used. These two ponds are surrounded by other farms and drain their water to
the sea, which is also the source of water. Bacteria in the seawater are therefore exposed
to different antimicrobials, which may result to cross-resistance between other aquacul-
ture antibacterials. The high incidence of antibiotic resistance in bacteria from the
incoming water of ponds currently using OXA and ponds that have previously used
antimicrobials may have contributed to the high antibiotic resistance in bacteria from
these ponds. Results show that the use of chemotherapeutants has a long-lasting effect
on the microflora of the receiving environment, which thus must be avoided.
The presence of antibiotic-resistant bacteria from ponds that did not use any
antimicrobial may be attributed to the gut flora of the cultured shrimp which may have
acquired resistance to certain antimicrobials during the hatchery phase of its culture.
Baticados and Paclibare Ž1992. reported that a large array of drugs is applied in shrimp
hatcheries. Terrestrial bacteria entering into the seawater with antibiotic-resistant plas-
mids may also be responsible for the prevalence of resistance genes in the marine
environment ŽChandrasekaran et al., 1998.. Exposure to heavy metals and other toxi-
cants can confer resistance to some antibiotics as well ŽTimoney and Port, 1982; Baya et
al., 1986.. The presence of antibiotic-resistant bacteria from ponds that did not use any
antimicrobial warrants further study.
Higher percentage of bacteria from pond water and soil than from the receiving water
and soil of all the three pond types were sensitive to the four tested antimicrobials. The
pond goes through pond preparation, which involves a series of flushing, tilling, drying
and liming, thereby killing most of the bacteria present. Exposure to sunlight may also
degrade some antimicrobials, like OTC ŽOka et al., 1989., if present.
Incidence of resistance to OTC was highest among individual antibiotics. OTC is
more extensively used in the Philippine aquaculture industry and was the first to be
available in the local market. There have also been reports that increased frequencies of
strains resistant to OTC can occur in an environment that had not used the antimicrobial
ŽKapetanaki et al., 1995; Vaughan et al., 1996; McPhearson et al., 1991.. Lunestad and
Goskøyr Ž1990. also reported that OTC-resistant bacteria are common. Oxytetracycline
belongs to the tetracycline group of antibiotics, which have a broad spectrum of activity
against many gram-positive and gram-negative bacteria. Tetracycline resistance is due to
three different mechanisms, which are all plasmid-mediated. Lewin Ž1992. suggested
that plasmids carried by fish pathogens may potentially serve as a reservoir for
resistance to other antimicrobials.
Incidence of resistance to furazolidone was observed next to resistance to oxytetracy-
cline. Furazolidone is the only nitrofurans currently permitted for use in food-producing
species. Nitrofurans are synthetic antibacterial compounds and are very effective drugs.
Oka et al. Ž1995. reported that they do not appreciably contribute to the development of
resistance, which is in contrast with this study.
Incidence of resistance to chloramphenicol was the least observed among the
individual antibiotics. This conforms to the observation of Baticados et al. Ž1990., Leano ˜
202 ˜ r Aquaculture 195 (2001) 193–204
E.A. Tendencia, L.D. de la Pena

et al. Ž1999. and Abraham et al. Ž1997.. Abraham et al. Ž1997. observed that all isolates
from diseased shrimp were sensitive to chloramphenicol. Leano ˜ et al. Ž1999. reported
that only 2.2% of the luminous Vibrio they isolated were resistant to chloramphenicol.
In addition, the Philippine government in 1990 banned the use of chloramphenicol on
animals intended for human consumption ŽADBrNACA, 1991.. However, a survey
conducted by Primavera et al. Ž1993. documented that 4 out of 21 farmers interviewed
were using the drug. Chloramphenicol is a synthetic broad-spectrum antibiotics whose
resistance is due to a single plasmid-mediated mechanism.
Resistance of an isolate to both oxolinic acid and furazolidone was highest compared
to other antibiotic resistance profiles. This result agrees with Nygaard et al. Ž1992. who
observed higher frequency resistance to furazolidone in sediments with oxolinic acid
than in sediments with OTC. The same study reported that exposure to oxolinic acid
initiates resistance to other drugs to a larger extent than exposure to OTC. In gram-nega-
tive bacteria, resistance to the 4-quinolones Ži.e., oxolinic acid. is due to reduced drug
accumulation, which is often due to alterations in outer membrane proteins and is often
associated with cross-resistance to antibacterials that are unrelated to the 4-quinolones
ŽLewin, 1992..
Variations in the resistance to the different antimicrobials observed between two
ponds within the same group in this study may be attributed to the locality and the
source of prawn fry that were stocked.
Results of the study have established that multiple antibiotic resistance ŽMAR. and
intermediate sensitivity test reaction to one chemotherapeutant, which may result to
drug-resistant bacterial strains and affect future therapy of shrimps as well as human
diseases, are associated with antimicrobial use thus, the use of antibiotics in aquaculture
even at low concentration should be avoided.

Acknowledgements

The authors wish to thank the Aquaculture Department of the Southeast Asian
Fisheries Development Center for funding the study under study code FH 02 C97T; the
shrimp farmers—Atty. Eduardo Azarraga, Mr. Emil Bautista, Mr. Joven Chavez, Mr.
Stephen Lim, Dr. Manual Lim, and Mr. Ivan Yaptangco; and the Fish Health Section
˜
support staff—Helen Marcial, Mila Paner, Maila Sobejano, and Remia Travina.

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