Shrimp cultivation and export in Bangladesh have undergone
rapid expansion over the last two decades. Between 1983 and
2003, the volume of shrimp and prawn cultivated in land
has increased more than 14 times1. In 2003, the Department of
Fisheries estimated that there had been approximately 203,071
hectares of coastal shrimp farms producing an average of 75,167
metric tons of shrimp annually1. Export of shrimps has given
second priority in terms of earning foreign currency after the
Currently, 36 shrimp species are harvested and cultivated in
Bangladesh. The two main species of shrimps are the bagda
(shrimp) and the golda (prawn). Chittagong-Cox’s Bazar and
Khulna-Shatkira-Bagherat regions usually dominate shrimp
production. These two regions account for approximately 95
percent of the total area dedicated to shrimp culture. Brackish
water aquaculture is widespread throughout Satkhira, Khulna,
Cox’s Bazar, and Bagerhat. Penaeus monodon and
Macrobrachium rosenbergii, the two major species of bagda
and golda, respectively, are cultivated in these areas.
Macrobrachium rosenbergii, the most popular freshwater
prawn, is largely produced in the southwest region of the country.
There has been an increase in awareness about the nutritional
value and health benefits of fish consumption in the last two
Bangladesh J Microbiol, Volume 29, Number 1, June 2012, pp 7-10
decades. On the other hand, seafood is also known to have been
responsible for a significant percentage of food-borne diseases2,
3. In the seafood related outbreaks, a wide variety of viruses,
bacteria, and parasites have been reported. Consumption of raw
or undercooked seafood is the factor most commonly
associated with infection4. There is epidemiological evidence,
particularly from Japan, that consumption of raw fish is indeed
the cause of many outbreaks of food-borne diseases. Biotoxins
and histamines make up a large proportion of these outbreaks5.
Though viruses are the most common cause of seafood related
infections, most of the hospitalisation and deaths are due to
bacterial agents4. As a consequence, food safety and quality
aspects in trade became important, since fresh food is more
prone to microbiological contamination6. Therefore, with the
growing importance of shrimp as one of the major export items
from Bangladesh, it is important to carefully maintain the
microbiological quality of the exported item through appropriate
In this perspective, we attempted to monitor the pathogenic load
randomly in export quality shrimps. However, microbiological
proliferation in the shrimp is mainly dependent on the condition
of transport, handling and processing. Frozen shrimps are often
contaminated after catching7. Handling of raw materials
influences the bacteriological quality of frozen shrimps.
Insufficiently iced and improperly storage of shrimps at higher
temperature enhances the growth of microorganisms
Prevalence of Pathogenic Bacteria in Shrimp Samples Collected from
Hatchery, Local Markets and the Shrimp Processing Plant
M. Majibur Rahman, Farjana Rahman, Farzana Afroze, Farzana Yesmin, Kazi Kaniz Fatema, Kamal Kanta
Das, Rashed Noor*
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka-1217, Bangladesh.
Export quality frozen shrimps comprise as one of the major economic interests in Bangladesh. During cultivation
or processing and packaging, microbial condition is an important issue to maintain the quality of shrimps. Eight
shrimp samples, of which, one from hatchery, 3 from local markets and 4 processed export quality samples were
studied for microbiological risk assessment (MRA). One hatchery, three market and two export quality shrimp
samples were found to contain bacterial pathogens probationary identified as Escherichia coli, Klebsiella spp., Vibrio
spp., Aeromonas spp., Pseudomonas spp., Listeria spp., Shigella-like organisms, Staphylococcus aureus , and Salmonella
spp. and two other processed export quality shrimp samples were completely free of pathogens of any type.
Surprisingly, the later two processed export quality shrimp samples showed antibacterial activity against E. coli,
Klebsiella sp. and Vibrio sp.
Key Words: Shrimp; pathogenic microorganisms; export quality frozen shrimp, antimicrobial activity
Dr. Rashed Noor, Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka-1217,Tel: 8355626, 8355596 ext. 472, Cell: +8801749401451, E-mail:
responsible for microbiological changes. Thus, the quality of
shrimp and frozen fish of desirable food standards has to be
ensured with necessary measures. Frozen shrimps are normally
subjected to preshipment inspection based on physical and
sensory characteristics followed by microbiological
characteristics. Thus, our study was designed to i) identify and
enumerate the pathogens associated with the shrimps dedicated
for export, with the shrimps from hatchery and from local
markets; ii) to compare the microbiological data among the
various shrimp samples studied; and iii) finally to assess whether
is any residual antimicrobial agents left in the sample even after
Materials and Methods
Export quality frozen shrimp samples were collected from
Conception Sea Food Ltd., Cox’s Bazar and Seamark (BD)
Limited, Chittagong, Bangladesh. Hatchery shrimp was
collected from Shatkhira, Khulna. Market shrimps were
collected from Malibagh bazaar, Shantinagar market and Agora
departmental store. One fresh (Macrobrachium rosenbergi,
sample code ES 4), 2 sea water (Penaeus monodon, sample
code ES 1, 2 & 3) export quality frozen shrimp samples, 1
hatchery and 3 market shrimp samples were subjected to
Ten grams of samples from different parts (head, body and head
body) of each shrimp were aseptically collected and were
transferred to sterile containers. The sample was transferred to
90 ml sterile normal saline and was homogenized. Then 1 ml of
the homogenized suspension was transferred to 9 ml normal
saline and then serial dilution (10-fold) was carried out up to
10 6 consecutively.
Isolation of pathogenic microorganisms from shrimp sample
Escherichia coli and Klebsiella spp.
To isolate Escherichia coli and Klebsiella spp. from each 10-1
to 10-3 dilution tubes, 0.1 ml suspension was spread on the
surface of MacConkey agar medium and was incubated at 37
°C for 24 h. After incubation, the plate was observed for
characteristic colonies. The presence of E. coli was further
confirmed by the appearance of bluish-black colonies with
green metallic sheen on Eosin-Methylene Blue (EMB) agar
Salmonella spp., Shigella-like organisms and Vibrio spp.
One ml of homogenized sample was transferred to 9ml of
selenite cystine broth and alkaline peptone water (10-1 dilution)
for enrichment of Salmonella, Shigella like organisms and
Vibrio spp., respectively, which were then incubated at 37 °C
for 6 h. Then 1 ml of enriched broth was subjected to 10-fold
serial dilution up to 10-2 to 10-6 in 9 ml of normal saline. From
each 10-4 to 10-6 dilution tubes, 0.1 ml of suspension was spread
onto XLD and TCBS agar plates. After incubated at 37 °C for
24 h, characteristic colonies were detected and counted.
Each sample was mixed in sterile saline in a ratio of 1:8 and
was heated at 80 °C for 15 minutes in order to kill vegetative
cells. Then 1 ml heated suspension was allowed to grow at 37°
C in 9 ml fluid thioglycolate broth for 4 hrs. Afterward, 1 ml of
enriched broth was subjected to 10-fold serial dilution from
10 1 to 10-6 in 9 ml of normal saline. From each 10-4 to 10-6
dilution tubes, 0.1 ml of suspension was pour plated on
perfringens agar medium. The plates were then incubated at 37
°C in a candle jar for 48 hrs. Colonies appeared as black were
detected and counted as suspected Clostridium perfringens.
To isolate Listeria monocytogenes from 10-3, 10-5, 10-6 dilution
tubes, 0.1 ml suspension was spread onto Listeria isolation
media and was incubated at 37 ºC for 24 h. Colonies appeared
as olive green were detected and counted as suspected Listeria
Probationary identification of the bacterial isolates
Different types of colonies in various culture media were
observed carefully. Morphological characteristics including
color, shape, elevation, surface texture, opacity, etc of the
colonies on different media were recorded. The size and shape
of the cells were observed by Gram staining. Finally, according
to the Manual of Methods for General Bacteriology (ASM,
1981), a series of biochemical tests were performed to
probationary identify the bacteria of interest.
Tests for antimicrobial activity
Muller Hinton agar was seeded with test organisms (E. coli,
Klebsiella spp. and Vibrio spp.). Sterile paper discs (6.0 mm)
were impregnated each with 10 µl of homogenized shrimp
samples and were placed on the seeded agar plates. Plates were
then incubated at 37 oC for 24 h, and the clear zones of inhibition
(in mm) around the disc were measured with the slide calipers
and the results were recorded.
Microbiological assessment of different samples (hatchery,
local market and processed export quality)
Presence of pathogenic microorganisms was examined through
the conventional cultural and biochemical methods. Various
types of selective media that encourage the growth of specific
microorganisms were also used. XLD agar was used for
Salmonella and Shigella, TCBS agar for Vibrio spp. and MSA
agar media was used for Staphylococcus spp. In XLD agar media,
black centered colonies were observed that were assumed as
Rahman et al.
Salmonella spp. In TCBS agar media green and yellow colonies
were observed. Yellow colonies were suspected as Vibrio
cholerae and green colonies were suspected as Aeromonas and
Vibrio parahaemolyticus as it had no ability to ferment sucrose
in the TCBS agar. Olive green colonies with black center hollow
were identified as Listeria spp. on Listeria identification agar
media. Colonies from different selective media were
subcultured and subjected to extensive biochemical test for
confirmation. Results are given in the Table 1.
Comparison among results of pathogenic bacteria isolated from
local market, hatchery and processed export quality frozen
A comparative study was carried out to assess the quality of
supplied shrimp samples collected from local markets, hatchery
and shrimp industries. Pathogenic microorganisms associated
with health hazards were probationary identified as E. coli,
Klebsiella spp., Vibrio spp., Listeria spp., Salmonella spp. and
Staphylococcus aureus. The results are summarized in Tables
1 & 2. A number of bacterial pathogens were found from shrimp
samples collected from different local markets and hatchery.
No pathogens were detected from processed export quality
frozen shrimp in samples 1 and 2. The higher pathogenic
bacterial load was observed in case of processed export quality
frozen shrimp samples 3 and 4.
Tests for antimicrobial activity
The complete absence of pathogens led us to assume that the
two processed export quality shrimp (ES1 & 2) samples might
consist of some chemicals inhibitory against pathogens. Thus,
we turned our interest to examine whether the sample contained
any antimicrobial agent. Thus, the shrimp samples were tested
against three common test organisms (E. coli, Klebsiella spp.
and Vibrio spp.). Complete zone of inhibition was observed
around test organisms after introduction of processed export
quality shrimp samples but no zone of inhibition was found in
case of hatchery shrimp (Table 3).
Table 1. Biochemical tests confirmative of the presence of the specific pathogens
Colonies on media TSI Catalase Oxidase Indole MR VP Citrate Suspected organism
Slant Butt Gas H2S test test production Utilization
Mac(Pink) A A + - + - + + - + E. coli
MacConky(Gummy) A A - - + + - - - - Klebsiella spp.
Listeria media (black) - - - - - - - - - - Listeria monocytogenes
TCBS (Large Yellow) A A + - + - + + - + Aeromonas spp.
TCBS(Small Yellow) A A - - + - + + - + Vibrio cholerae
TSI: Triple Sugar Iron A: Acidic Reaction
MR: Methyl Red K: Alkaline reaction
VP: Voges Praskeur + : Positive
- : Negative
Table 2. Comparison among the pathogenic load of the shrimp samples from market, hatchery and from processed export
quality frozen shrimps
Isolated Pathogenic Bacteria Count (cfu/g)
MB SM ADS ES-1 ES-2 ES-3 ES-4 HS
E. coli 8x1051.20x1061.00x1050 0 1x1041x1041.2x107
Kliebsiella spp 1x1050 0 0 0 4x1046.5x1052.0x107
Vibrio spp. 1.15x1061.26x1076.50x1050 0 3x1044.5x1052.9x105
Aeromonas spp. 0 0 0 0 0 0 0 2.7x105
Pseudomonas Spp. 0 0 0 0 0 0 0 5.5x107
Shigella like organisms 3.00x1062.00x1051.02x10700 0 0 0
Salmonella spp. 0 0 0 0 0 0 2x1040
Clostridium spp. 0 0 0 0 0 0 0 0
Staphylococcus aureus 4.58x1076.57x1072.09x1070 0 1.50x1071.01x1070
Listeria spp. 0 7.85x1070 0 0 3x1044.5x1053x107
MB: Malibagh Bazar SM: Shantinagar Market
ADS: Agora Departmental Store ES: Export Quality Frozen Shrimp
HS: Hatchery shrimp
Prevalence of Pathogenic Bacteria in Shrimp Samples Collected
Table 3. Antimicrobial activity exhibited by export quality
Sample code Zone of Inhibition (mm) against pathogens
E. coli Klebsiella Vibrio
HS-1 Nil Nil Nil
ES-1 24 18 15
ES-2 19 17 12
HS: Hatchery shrimp
ES: Export quality frozen shrimp Nil: no zone of inhibition found around
the sample used
Fisheries sector plays an important role in the socio-economic
development of Bangladesh. This is the second highest source
of earning foreign currencies and providing direct or indirect
employment to 10% of the total population of the country1.
The quality of the processed products largely depends on the
quality of raw materials and it is difficult to preserve freshness
of raw materials when there is a long gap between the harvesting
and processing time. During this period, shrimps continue to
deteriorate8. The time interval between the landings of shrimps
and their arrival at the processing plants is very important9.
Improper handling and inadequate processing result in microbial
growth which causes spoilage of food products. Present study
was thus carried out to assess the pathogenic load in the shrimp
samples of export quality.
Bangladesh exports mainly frozen shrimp and different types
of fresh and marine water fishes. The recent introduction of the
Hazard Analysis Critical Control Point (HACCP) system and
European Union hygienic regulations in seafood industries will
pave the way for the production of safe and high quality seafood.
Bangladesh frozen shrimp exporters continue to have both real
and perceived problems with buyers in the USA, the European
Union and Japan, concerning the safety and quality of their
products. Because many fish processing plants in Bangladesh
do not follow the HACCP system and EU hygienic regulations
for which many of them have been banned and the export of
shrimps, fish and fish products is reduced10. From the standpoint
of above observations, we have tried to assess the quality of our
shrimp products and our study detected the presence of
pathogens (Escherichia coli, Klebsiella spp., Vibrio cholerae,
Listeria monocytogenes) associated with enteric diseases in
human from shrimp sample collected from hatchery and market
which exceed the standard level of ICMSF (International
Commission on Microbial Specifications for Foods). Pathogens
were also identified from export quality shrimp samples which
may occur due to inadequate processing and transportation. Thus,
the presence of pathogens in shrimp samples suggests the fact
that proper handling and maintenance is necessary. Another
interesting finding of our study reveals that chemical
preservatives or antibiotics may be used by the shrimp
processing industries to maintain the standard quality. For
establishment of the significant validity of this test, further
chemical analysis will unveil the accuracy of shrimp processing
in future in Bangladesh.
Antimicrobial agents are being used worldwide in aquaculture
to present infection. The commonly used antibiotics are
sulphonamides, tetracycline, amoxicillin, trimethoprim-
sulphamethoxine and quinolones11. Our results also showed the
presence of antimicrobial agents in shrimp samples. However,
extensive biological and chemical characterization is required
to confirm the presence of a particular agent and its
concentration as high concentration of preservative of any type
is health hazardous.
Overall, according to the present study, shrimps of hatcheries
and local markets have been found to harbor many pathogens
indicating that these are not protected from contamination during
subsequent handling, packaging, storage, and transport.
Microbiological quality is a very vital aspect of quality control
measures; proper handling of fish between capture and delivery
to the consumer is a crucial element in assuring quality of the
final product. Standard sanitation, handling and the time &
temperature of holding fish are significant factors to ensure
quality. Properly controlled monitoring is also required for
transportation and storage. If HACCP guidelines are strictly
followed during processing and handling, the risk of
contamination and outbreak of diseases would be minimized.
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Department of Fisheries, Ministry of Fisheries and Livestock, Government
of the Peoples Republic of Bangladesh.
2. Karunasagar I, Pai R, Malathi GR and Karunasagar I. 1994. Mass mortality
of Penaeus monodon larvae due to antibiotic resistant Vibrio harveyi
infection. Aquaculture 128: 203-209.
3. Wallace BJ, Guzewich JJ, Cambridge M, Altekruse S and Morse DL. 1999.
Seafood-Associated Disease outbreaks in New York, 1980-1994. American
J Preventive Med. 17 (1): 48-54.
4. Butt AA, Aldridge KE and Sanders CV. 2004. Infections related to the
ingestion of seafood Part I: viral and bacterial infections. The Lancet Infect
Dis. 4: 201-212.
5. Huss HH. 1997. Control of indigenous pathogenic bacteria in seafood.
Food Control. 8(2): 91-98.
6. Sawhney A. 2005. Quality Measures in Food Trade: The Indian Experience.
The World Economy. 28 (3): 329-348.
7. Robinson RK. 1985. In Microbiology of Frozen Food. Elsevier Applied
8. Antony MM, Jeyasekaran G, Shakila RJ and Shanmugam SA. 2002.
Microbiological Quality of Raw Shrimps Processed in Seafood Processing
Plants of Tuticorin, Tamil Nadu, India. Asian Fish Sci. 15: 33-41.
9. Rajadurai NP. 1985. Improving the quality of shrimp through proper
handling. INFOFISH International. 1: 50-52.
10. CFC/INFOFISH. 1997. CFC/INFOFISH Project on Export Promotion of Value-
Added Fishery Products and their Sustainable Development. Progress
Report (1 January – 30 June 1997). Kuala Lumpur, Malaysia: INFOFISH.
11. Angulo F. 1999. Use of antimicrobial agents in aquaculture: potential for
public health impact. Division of Bacterial and Mycotic Diseases. Center
for Disease Control and Prevention. Accessed 24 August, 2007.
Rahman et al.