2.1 Shrimps; Species of study, Shrimps Industry, Microbial Contamination.


2.1       Shrimps; Species of study, Shrimps
Industry, Microbial Contamination.

2.1.1    White leg shrimp, Litopenaeus vannamei

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            White leg shrimp, Litopenaeus
vannamei or can be recognized as Penaeus vannamei is one of the
important shrimp species in farmed worldwide especially in developing country.

Besides that, it is also commonly recognized as Mexican white and Pacific
white. White leg shrimp can be found widely where water temperatures are
normally more than 20°C
but the growth is much higher at 30°C
and it is native to the Eastern Pacific coast from Sonora, North Mexico,
Central and South America as well as Tumbes in Peru. White leg shrimp lives in
tropical marine habitats which suits the environment in Malaysia. L.

vannamei is omnivorous organism that likes muddy bottoms and can feed
efficiently compared to tiger prawn. L. vannamei has bluish hue and
translucent in white colour and thus it is called white leg shrimp. Adult white
leg shrimp live and spawn in the open ocean meanwhile post larvae will move to
inshore then adolescent and sub-adult stay in coastal estuaries.


            The production of this species took
53.1% of total production of crustaceans in aquaculture world. L. vannamei was
produced at 98.6% in developing countries in 2014 (Qiu et al. 2017). This
species is one of the major export in Malaysia and thus it is crucial that this
species to be studied effectively.


2.1.2    Giant Tiger Prawn, Penaeus monodon

            Giant Tiger Prawn, P. monodon or
it is recognized as Asian Tiger Shrimp belongs to the family Penaeidae
(Fuller et al.,2014). They are native to the Indo-West Pacific including East
Africa, South and Southeast Asia, the Philippines and Australia (Sandoval,
Leal-Florez, Taborda & Vásquez, 2014; Li et al. 2016). The growth of P.

monodon takes place in estuaries, coastal lagoons and mangroves when they
are at larval, juvenile and sub-adult stages respectively. Meanwhile, adult
stages will inhabit the continental shelf and then upon maturation, they will
reproduce in tropical marine habitats (Sandoval et al., 2014). Li (2016) stated
that the growth of this organism depends on the sex, developmental stage and
also environment. This species is established in Malaysia and many other areas
due to escapes from aquaculture which includes West Africa, the Caribbean and
from the northern and north-eastern of South America from Venezuela to eastern
Brazil. This species dominated shrimp industry due to its high growth rate,
highly nutritive, tasty and constant demand in global market (Mohanty, Mishra
& Patil, 2014)


2.1.3    Shrimp Industry

            In Malaysia, marine prawn P.

monodon and P. vannamei are the main species cultured. Aquaculture
industry in Malaysia has grown rapidly in this decade especially when the
government emphasize the role of aquaculture sector in Third National
Agriculture Policy (1998-2008) followed with National Agro-Food Policy
(2011-2020). Aquaculture industry in Malaysia has started since 1920’s which
was started with freshwater aquaculture and brackish-water aquaculture in 1930s
whereas Sabah and Sarawak started in the early 1990s (Hamdan, Othman &
Kari, 2015). It becomes a huge industry due to the location of Malaysia which
is surrounded by South China Sea and the Strait of Malacca. Besides that, the
government has encouraged and give incentives to aquaculture industry (Yusoff,
2015; Ghee-Thean, Islam, Ismail, 2016; Hamdan et al., 2015). One of the factor
that shrimp industry has become global and domestic demand is due to change of
preference from red meat to white meat such as shrimp and fish product
(Ghee-Thean et al., 2016).


2.1.4    Microbial Contamination in Shrimps

            Animal protein such
as shrimp is a high risk commodity when it comes to pathogenic bacteria
contamination and it may also contain natural toxins and adulterants (Mandal et
al., 2015; Muhammad-Yousuf et al., 2008). Shrimp can be contaminated with water
and sediments which contains naturally found pathogen such as Vibrio
spp., non-typhi Salmonella and Campylobacter (Elbashir et al.,
2018). According to CDC (2014), there are several etiologic agents that can
cause seafood-borne illnesses which are norovirus, Vibrio, Salmonella,
E. coli, Campylobacter, Shigella, C. botulinum and Staphylococcus
aureus (as cited in Elbashir et al., 2018). In addition, shrimp may also be
contaminated with coliform, Salmonella spp., Staphylococcus spp.

and other food borne bacteria (Ghalib et al., 2014).


            Possible route of contamination may
take place by direct contact, from the airborne particles and by the attachment
on the product surface. Some pathogenic bacteria are capable to attach to the
food surfaces as their predominant form of survival when stress in introduced.

Bacterial contamination in shrimp can also happen due to poor hygienic
condition during processing and storage condition. Shrimp can be contaminated
with Vibrio spp. which is Gram-negative bacteria that naturally lives in
estuarine and marine environment (Letchumanan, Yin, Lee and Chan, 2015).


2.2       Escherichia
coli; natural habitat, E. coli in Shrimps.

2.2.1    Natural habitat of E. coli

            Escherichia coli is
a facultative anaerobe gram negative bacteria from bacterial family of
Enterobacteriaceae which is non-spore forming organism and rod-shaped with
flagella. E. coli can be found in intestine of humans and animals. E.

coli habitat can be classified into two types which are primary and
secondary habitat. Primary habitat of E. coli is in living host like
animal and human which is in the gastrointestinal tract of mammal whereas
secondary habitat is where E. coli can be found in open and non-host
environment such as soil and water (Gordon, Bauer & Johnson, 2002).

Ghaderpour et al. (2015) said that E. coli is common in the intestine of
humans and warm-blooded animals where it can also be an indicator of fecal
contamination in aquatic environment and food. Dutta and Sengupta (2016) also
stated that E. coli can be an indicator organism to determine fecal
contamination of water and seafood.


            According to Atnafie et al. (2017),
not all types of E. coli are
pathogenic to human but there are some strains of E. coli that can cause adverse effects such as shiga
toxin-producing E. coli O157:H7 (STEC
O157). Ingestion of E. coli O157:H7
can cause abdominal pain, haemorrhagic colitis, haemolytic uremic syndrome
(HUS) and bloody diarrheal which can lead to kidney failure (Atnafie et al.,
2017; Kumaran, Deivasigamani, Alagappan, Sakthivel & Karthikeyan, 2010).

Kumaran et al. (2010) also mentioned that adverse effects resulted from E. coli O157:H7 is life threatening and
the effect is more significant in young kids and elderly.


2.2.2    Occurrence of Escherichia coli in

            E. coli is not naturally
found in aquatic organism but it can be the indicator of environment
contamination on shrimps due to its abundance in water (Mandal et al., 2015).

Ghaderpour et al. (2015) state that B1 phylogenetic group of E. coli is
able to endure the stress in aquatic environment compared to A, B2 and D
groups. According to Dutta and Sengupta (2016), findings of their study showed
that there were a huge percentage of prevalence of E. coli which is 85%
from the sample taken. Besides that, they stated that contamination of this
bacteria is quite common in India due to partially treated or untreated sewage
water where E. coli had been isolated from coastal beach and seawater.

Coastal areas or estuaries has often become the location of aquaculture
business even though the level of water pollution from human and animal feces
are high (Boss, Overesch & Baumgartner, 2016). In addition, presence of E.

coli on aquatic organism reflect the hygiene of the farm and water also the
environment throughout the transportation until it reaches to customers.

Insufficiently treated water and even improper hygienic measures during
handling after catching and processing may cause growth of E. coli in
shrimps. It has been said by Dutta and Sengupta (2016), seafood quality is
defined by the quality of water from where the aquatic animal is harvested and
the environmental condition of the landing centers.


            Contamination of E. coli may also be due to handling
method and storage (Chakravarty,
Ganesh, Amaranth, Sudha & Subhashini, 2015). Even though E.

coli is abundance in water, it cannot thrive in marine environment for long
period of time (Dutta & Sengupta, 2016). The detection of this
microorganism might represent the contamination that occurs after harvest.

According to Mandal et al. (2015), there were studies that indicate
contamination of food borne bacteria at the equipment and utensil used in
processing. These bacteria such as E. coli, S. aureus and Salmonella spp.

can survive on the surfaces of hands and utensil after the initial contact.

Samsul, Adamu, Mohd Desa and Khairani-Bejo (2016) found that hand washing as
hygiene practices was not practiced by workers in some locations especially
after work. In addition, E. coli has been detected from water vending
machines that could be due to cross contamination that happens result in lack
of hygienic practices (Tan, Arifullah & Soon, 2016).


2.3       S.

aureus; natural habitat, S. aureus in Shrimps.

2.3.1    Natural Habitat of S. aureus

aureus is a cocci shaped gram positive bacteria which is
non-motile, non-spore forming, capable of producing enterotoxin and it is
facultative anaerobe bacteria (Palilu & Budiarso, 2017). S. aureus does
not require any specific requirements and conditions to grow because it is
able to grow in low water activity as low as 0.86, pH value ranging from 4.2 to
9.3 and capable of growing from 7 to 48°C
(Arfatahery, Mirshafiey, Abedimohtasab & Zeinolabedinizamani, 2015; Palilu
& Budiarso, 2017). S. aureus has been recognized as one of the most
pathogen that can cause food poisoning (Arfatahery, Davoodabadi &
Abedimohtasab, 2015). It can be found easily in natural environment which it is
commonly associated with colonization of skin and mucosal surfaces of humans
and animals (Kumar et al., 2016).


            Ingestion of S. aureus can
cause food poisoning which happens when food is contaminated with enterotoxin
or due to the ingestion of the bacteria itself. S. aureus also has been
responsible for hospital-acquired infection (HAI) or nosocomial infections and
community acquired disease (Kumar et al., 2016). The most vulnerable group of
people are the elderly, infants, children, pregnant women, immunocompromised
patients, patients who take immune suppressing drugs and patients who are
undergoing tumor treatment (Arfatahery et al., 2015; Rong et al., 2017).


2.3.2    Occurrence S. aureus in Shrimp

S. aureus may contaminate food when handling
practices are not adequately practiced which occurs due to spread of this
pathogen from the processing equipment and food handlers to the food system
(Macedo, Marinho, Vasconcelos & Costa, 2016; Rong et al., 2017). According
to Arfatahery et al. (2015), studies were carried out previously in order to
determine the path of transmission of S. aureus. The findings showed
that it is spread by human carriers or environmental condition during
processing which includes transportation, packaging, cross-contamination of
workers’ hands and infected respiratory secretion that may be in contact with
the food or environment. Kumar et al. (2016) has suggested that presence of
this bacteria can be a reflection of contamination that could happen during
transport and handling which is introduced by the workers.


2.4       Antibiotic; Antibiotic in Animals,
Antibiotic Resistance.

            Antibiotic can be
defined as medicines that are used to prevent and treat bacterial infection. It
has been an effective way to treat infection of bacteria, invasive surgery,
cancer chemotherapy and also immune-compromised and elderly patients. Hughes
and Karlén (2014) mentioned that success of antibiotic as medical drugs is due
to its effectiveness in treating microbial infections rapidly, it is safe to
use and cost effective drugs.   


            Successful discovery of antibiotic
is started when Selman Walksman introduced a discovery platform that uses
soil-derived streptomycetes to determine microbial activity towards a test
microorganism by detecting zones of growth inhibition on an overlay plate
(Lewis, 2013). The method is similar with the discovery of penicillin by Sir
Alexander Fleming. In 1928, Sir Alexander Fleming has started the modern era of
antibiotic with the discovery of penicillin where it has been used to control
bacterial infections among soldiers in World War II. Fleming’s screening method
had been used by many researchers because it required less testing on animal
disease model but only requires agar-medium plates to determine the inhibition
of pathogenic bacteria. Another important individual that has been associate
with the beginning of modern antibiotic era is Paul Ehrlich. Ehrlich proposed
an idea which he focused on the disease-causing microorganism and not the host.

He also introduced a systematic screening approach which has become the
foundation of drug strategies in pharmaceutical industry. This approach leads
to thousands of drugs identification and becomes a clinical practice which
provides a variety of antimicrobial drugs. Nowadays, there are a lot of
antibiotic has been introduced which discovered from period of time from 1950s
to 1970s, which is recognized as the golden era of discovery of novel
antibiotic classes.


2.4.1    Antibiotic in animal

of antibiotic is not restricted to medical uses only but it also used in
non-medical purpose. Antibiotic has been used in animals for quite a long time
to accelerate the growth and to increase the size of the animals. Meek, Vyas
and Piddock (2017) mentioned that in United State, there are more antibiotics
used for animal growth compared to its usage in human medicine. Hughes and
Heritage (2004) stated that antibiotic growth promoter is registered in low
dosage to inhibit infection of microorganism (as cited in Ronquillo &
Hernandez, 2017). The use of antibiotic in animals such as chickens and pigs as
growth promoter has started in 1940s when chickens were fed with feed that
contains by-product of tetracycline fermentation whereas pigs’ feed were added
with dried mycelia of Streptomyces aureofaciens which contained
chlortetracycline residues (Ronquillo & Hernandez, 2017).


Antibiotic has also been used as veterinary drugs which functioned to
treat sickness in animals, confine the disease from spreading out of the farm
and prevent infections. Sekyere (2014) stated that disease in animals may
reduce productivity because it slows growth rate and increase mortalities;
therefore, farmers use doses of antibiotic to prevent these from happening.

Antibiotic is used for prophylaxis and metaphylaxis. Both of these antibiotic is
used to prevent and control diseases in animals. Prophylaxis drugs are
administered to maintain and prevent disease which it is given animals that at
a risk of a disease outbreak meanwhile metaphylaxis is an early curative
treatment which administered to animals even though there are no clinical
symptoms when there is an outbreak of a disease (Bousquet-Mélou, 2010).


On the other hand, aquaculture scale farming often stressful to the
aquatic organism which may impair their immune systems and thus they are more
vulnerable to infections

. Antibiotic has been used in aquaculture as prophylactic, metaphylactic
and therapeutic purpose. Therapeutic drugs are commonly administered according
to the rules from regulatory agencies to aquatic organism in short period of
time by oral route (Romero, Feijoó & Navarrete, 2012). There are set of
rules from regulatory agencies in every country that must be complied including
the routes of administration, form of drugs, withdrawal time, species,
tolerance, concentration and limitation.  Similar to how antibiotic is
administered to the livestock animals, antibiotic that is given to aquatic
animals also administered as part of feed. However, there might be residues of
antibiotic that may not consume by the animals and remain in the water (Meek,
Vyas & Piddock, 2015). Aquatic animals may not metabolize antibiotic
effectively which will contain in their faeces. According to Burridge et al.

(2010), 75% of antibiotic fed to the aquatic animals ended up excreted in
water. He also reported that level of antibiotics are varies due to different
practices in every country however Defoird et al. (2011) had estimate the
amount of antibiotic in shrimp production in Thailand and Vietnam where both
countries showed a large variation in usage of antibiotic (as cited in Romero,
Feijoó & Navarrete, 2012).


2.4.2    Antibiotic resistance

According to Center for Disease Control and
Prevention (2017), antibiotic resistance refers to bacteria that unaffected by
the effect of antibiotics. It occurs when the bacteria evolve in a way to
reduce the effectiveness of the antimicrobial agents. Antibiotic resistance has
been world’s most pressing public health problems because it can be spread
easily through the consumption of food that has been contaminated with
antibiotic resistance bacteria. Aquaculture animals are the most affected by
antibiotic resistance bacteria due to wide usage of antibiotic in farming which
may be excreted in faeces or by the residual antibiotic that is not consumed by
the aquatic animals.

Chakravarty et al. (2015)
found that E. coli in shrimps’ sample
from Visakhapatnam Fishing Harbour showed 100% resistance to penicillin and
tetracycline meanwhile 50% of the sample was resistance to gentamicin,
nitrofurantoin and ampicillin. Meanwhile, Boss et al. (2016) found that 12% of E. coli isolated from shrimps were
resistant to ciprofloxacin and nalidixic acid. They also found that S. aureus isolates were resistant to aminoglycosides, ?-lactams and tetracycline. On
the other hand, Macedo et al. (2016) detected antibiotic resistance S. aureus in fresh marine shrimp which
indicates that shrimps may be a vehicle to spread antibiotic resistant S. aureus.

Due to steady occurrence of
antibiotic resistant bacteria, it is crucial to identify antibiotic resistant
bacteria as it will give a major impact towards treatment of foodborne illness
and at the same time contribute a better control of antimicrobial resistance.

Monitoring of antibiotic resistant bacteria with accordance to Clinical and
Laboratory Standard Institute (CLSI) can help in providing the information
needed for treatment of ill patients (CLSI, 2015).