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

 2.1       Shrimps; Species of study, ShrimpsIndustry, Microbial Contamination.2.1.1    White leg shrimp, Litopenaeus vannamei            White leg shrimp, Litopenaeusvannamei or can be recognized as Penaeus vannamei is one of theimportant shrimp species in farmed worldwide especially in developing country.Besides that, it is also commonly recognized as Mexican white and Pacificwhite.

White leg shrimp can be found widely where water temperatures arenormally more than 20°Cbut the growth is much higher at 30°Cand 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 intropical marine habitats which suits the environment in Malaysia. L.vannamei is omnivorous organism that likes muddy bottoms and can feedefficiently compared to tiger prawn. L. vannamei has bluish hue andtranslucent in white colour and thus it is called white leg shrimp. Adult whiteleg shrimp live and spawn in the open ocean meanwhile post larvae will move toinshore then adolescent and sub-adult stay in coastal estuaries.

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             The production of this species took53.1% of total production of crustaceans in aquaculture world. L. vannamei wasproduced at 98.6% in developing countries in 2014 (Qiu et al. 2017). Thisspecies is one of the major export in Malaysia and thus it is crucial that thisspecies to be studied effectively.

 2.1.2    Giant Tiger Prawn, Penaeus monodon            Giant Tiger Prawn, P. monodon orit is recognized as Asian Tiger Shrimp belongs to the family Penaeidae(Fuller et al.

,2014). They are native to the Indo-West Pacific including EastAfrica, 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 theyare at larval, juvenile and sub-adult stages respectively. Meanwhile, adultstages will inhabit the continental shelf and then upon maturation, they willreproduce in tropical marine habitats (Sandoval et al., 2014). Li (2016) statedthat the growth of this organism depends on the sex, developmental stage andalso environment. This species is established in Malaysia and many other areasdue to escapes from aquaculture which includes West Africa, the Caribbean andfrom the northern and north-eastern of South America from Venezuela to easternBrazil.

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. Aquacultureindustry in Malaysia has grown rapidly in this decade especially when thegovernment emphasize the role of aquaculture sector in Third NationalAgriculture Policy (1998-2008) followed with National Agro-Food Policy(2011-2020). Aquaculture industry in Malaysia has started since 1920’s whichwas started with freshwater aquaculture and brackish-water aquaculture in 1930swhereas Sabah and Sarawak started in the early 1990s (Hamdan, Othman , 2015). It becomes a huge industry due to the location of Malaysia whichis surrounded by South China Sea and the Strait of Malacca.

Besides that, thegovernment has encouraged and give incentives to aquaculture industry (Yusoff,2015; Ghee-Thean, Islam, Ismail, 2016; Hamdan et al., 2015). One of the factorthat shrimp industry has become global and domestic demand is due to change ofpreference 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 suchas shrimp is a high risk commodity when it comes to pathogenic bacteriacontamination and it may also contain natural toxins and adulterants (Mandal etal.

, 2015; Muhammad-Yousuf et al., 2008). Shrimp can be contaminated with waterand sediments which contains naturally found pathogen such as Vibriospp., non-typhi Salmonella and Campylobacter (Elbashir et al.,2018). According to CDC (2014), there are several etiologic agents that cancause seafood-borne illnesses which are norovirus, Vibrio, Salmonella,E. coli, Campylobacter, Shigella, C. botulinum and Staphylococcusaureus (as cited in Elbashir et al.

, 2018). In addition, shrimp may also becontaminated with coliform, Salmonella spp., Staphylococcus spp.and other food borne bacteria (Ghalib et al., 2014).                         Possible route of contamination maytake place by direct contact, from the airborne particles and by the attachmenton the product surface.

Some pathogenic bacteria are capable to attach to thefood surfaces as their predominant form of survival when stress in introduced.Bacterial contamination in shrimp can also happen due to poor hygieniccondition during processing and storage condition. Shrimp can be contaminatedwith Vibrio spp. which is Gram-negative bacteria that naturally lives inestuarine and marine environment (Letchumanan, Yin, Lee and Chan, 2015). 2.2       Escherichiacoli; natural habitat, E. coli in Shrimps.2.

2.1    Natural habitat of E. coli            Escherichia coli isa facultative anaerobe gram negative bacteria from bacterial family ofEnterobacteriaceae which is non-spore forming organism and rod-shaped withflagella. E.

coli can be found in intestine of humans and animals. E.coli habitat can be classified into two types which are primary andsecondary habitat. Primary habitat of E.

coli is in living host likeanimal and human which is in the gastrointestinal tract of mammal whereassecondary habitat is where E. coli can be found in open and non-hostenvironment such as soil and water (Gordon, Bauer & Johnson, 2002).Ghaderpour et al. (2015) said that E.

coli is common in the intestine ofhumans and warm-blooded animals where it can also be an indicator of fecalcontamination in aquatic environment and food. Dutta and Sengupta (2016) alsostated that E. coli can be an indicator organism to determine fecalcontamination of water and seafood.             According to Atnafie et al. (2017),not all types of E. coli arepathogenic to human but there are some strains of E. coli that can cause adverse effects such as shigatoxin-producing E. coli O157:H7 (STECO157).

Ingestion of E. coli O157:H7can 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 andthe effect is more significant in young kids and elderly. 2.2.2    Occurrence of Escherichia coli inshrimp            E. coli is not naturallyfound in aquatic organism but it can be the indicator of environmentcontamination on shrimps due to its abundance in water (Mandal et al., 2015).Ghaderpour et al.

(2015) state that B1 phylogenetic group of E. coli isable to endure the stress in aquatic environment compared to A, B2 and Dgroups. According to Dutta and Sengupta (2016), findings of their study showedthat there were a huge percentage of prevalence of E.

coli which is 85%from the sample taken. Besides that, they stated that contamination of thisbacteria is quite common in India due to partially treated or untreated sewagewater where E. coli had been isolated from coastal beach and seawater.Coastal areas or estuaries has often become the location of aquaculturebusiness even though the level of water pollution from human and animal fecesare high (Boss, Overesch & Baumgartner, 2016).

In addition, presence of E.coli on aquatic organism reflect the hygiene of the farm and water also theenvironment throughout the transportation until it reaches to customers.Insufficiently treated water and even improper hygienic measures duringhandling after catching and processing may cause growth of E. coli inshrimps. It has been said by Dutta and Sengupta (2016), seafood quality isdefined by the quality of water from where the aquatic animal is harvested andthe environmental condition of the landing centers.

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

coli is abundance in water, it cannot thrive in marine environment for longperiod of time (Dutta & Sengupta, 2016). The detection of thismicroorganism might represent the contamination that occurs after harvest.According to Mandal et al.

(2015), there were studies that indicatecontamination of food borne bacteria at the equipment and utensil used inprocessing. 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 ashygiene practices was not practiced by workers in some locations especiallyafter work. In addition, E. coli has been detected from water vendingmachines that could be due to cross contamination that happens result in lackof hygienic practices (Tan, Arifullah & Soon, 2016). 2.3       S.aureus; natural habitat, S.

aureus in Shrimps.2.3.1    Natural Habitat of S.

aureus            Staphylococcusaureus is a cocci shaped gram positive bacteria which isnon-motile, non-spore forming, capable of producing enterotoxin and it isfacultative anaerobe bacteria (Palilu & Budiarso, 2017). S. aureus doesnot require any specific requirements and conditions to grow because it isable to grow in low water activity as low as 0.

86, pH value ranging from 4.2 to9.3 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 mostpathogen that can cause food poisoning (Arfatahery, Davoodabadi , 2015). It can be found easily in natural environment which it iscommonly associated with colonization of skin and mucosal surfaces of humansand animals (Kumar et al., 2016).             Ingestion of S.

aureus cancause food poisoning which happens when food is contaminated with enterotoxinor due to the ingestion of the bacteria itself. S. aureus also has beenresponsible for hospital-acquired infection (HAI) or nosocomial infections andcommunity acquired disease (Kumar et al., 2016). The most vulnerable group ofpeople are the elderly, infants, children, pregnant women, immunocompromisedpatients, patients who take immune suppressing drugs and patients who areundergoing tumor treatment (Arfatahery et al., 2015; Rong et al., 2017). 2.

3.2    Occurrence S. aureus in ShrimpS. aureus may contaminate food when handlingpractices are not adequately practiced which occurs due to spread of thispathogen from the processing equipment and food handlers to the food system(Macedo, Marinho, Vasconcelos & Costa, 2016; Rong et al., 2017). Accordingto Arfatahery et al. (2015), studies were carried out previously in order todetermine the path of transmission of S. aureus.

The findings showedthat it is spread by human carriers or environmental condition duringprocessing which includes transportation, packaging, cross-contamination ofworkers’ hands and infected respiratory secretion that may be in contact withthe food or environment. Kumar et al. (2016) has suggested that presence ofthis bacteria can be a reflection of contamination that could happen duringtransport and handling which is introduced by the workers. 2.

4       Antibiotic; Antibiotic in Animals,Antibiotic Resistance.            Antibiotic can bedefined as medicines that are used to prevent and treat bacterial infection. Ithas been an effective way to treat infection of bacteria, invasive surgery,cancer chemotherapy and also immune-compromised and elderly patients. Hughesand Karlén (2014) mentioned that success of antibiotic as medical drugs is dueto its effectiveness in treating microbial infections rapidly, it is safe touse and cost effective drugs.                            Successful discovery of antibioticis started when Selman Walksman introduced a discovery platform that usessoil-derived streptomycetes to determine microbial activity towards a testmicroorganism by detecting zones of growth inhibition on an overlay plate(Lewis, 2013). The method is similar with the discovery of penicillin by SirAlexander Fleming. In 1928, Sir Alexander Fleming has started the modern era ofantibiotic with the discovery of penicillin where it has been used to controlbacterial infections among soldiers in World War II.

Fleming’s screening methodhad been used by many researchers because it required less testing on animaldisease model but only requires agar-medium plates to determine the inhibitionof pathogenic bacteria. Another important individual that has been associatewith the beginning of modern antibiotic era is Paul Ehrlich. Ehrlich proposedan idea which he focused on the disease-causing microorganism and not the host.He also introduced a systematic screening approach which has become thefoundation of drug strategies in pharmaceutical industry.

This approach leadsto thousands of drugs identification and becomes a clinical practice whichprovides a variety of antimicrobial drugs. Nowadays, there are a lot ofantibiotic has been introduced which discovered from period of time from 1950sto 1970s, which is recognized as the golden era of discovery of novelantibiotic classes. 2.4.1    Antibiotic in animal            Usageof antibiotic is not restricted to medical uses only but it also used innon-medical purpose. Antibiotic has been used in animals for quite a long timeto accelerate the growth and to increase the size of the animals. Meek, Vyasand Piddock (2017) mentioned that in United State, there are more antibioticsused for animal growth compared to its usage in human medicine.

Hughes andHeritage (2004) stated that antibiotic growth promoter is registered in lowdosage to inhibit infection of microorganism (as cited in Ronquillo , 2017). The use of antibiotic in animals such as chickens and pigs asgrowth promoter has started in 1940s when chickens were fed with feed thatcontains by-product of tetracycline fermentation whereas pigs’ feed were addedwith dried mycelia of Streptomyces aureofaciens which containedchlortetracycline residues (Ronquillo & Hernandez, 2017). Antibiotic has also been used as veterinary drugs which functioned totreat sickness in animals, confine the disease from spreading out of the farmand prevent infections. Sekyere (2014) stated that disease in animals mayreduce 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 isused to prevent and control diseases in animals.

Prophylaxis drugs areadministered to maintain and prevent disease which it is given animals that ata risk of a disease outbreak meanwhile metaphylaxis is an early curativetreatment which administered to animals even though there are no clinicalsymptoms when there is an outbreak of a disease (Bousquet-Mélou, 2010). On the other hand, aquaculture scale farming often stressful to theaquatic organism which may impair their immune systems and thus they are morevulnerable to infections. Antibiotic has been used in aquaculture as prophylactic, metaphylacticand therapeutic purpose. Therapeutic drugs are commonly administered accordingto the rules from regulatory agencies to aquatic organism in short period oftime by oral route (Romero, Feijoó & Navarrete, 2012).

There are set ofrules from regulatory agencies in every country that must be complied includingthe routes of administration, form of drugs, withdrawal time, species,tolerance, concentration and limitation.  Similar to how antibiotic isadministered to the livestock animals, antibiotic that is given to aquaticanimals also administered as part of feed. However, there might be residues ofantibiotic that may not consume by the animals and remain in the water (Meek,Vyas & Piddock, 2015). Aquatic animals may not metabolize antibioticeffectively which will contain in their faeces. According to Burridge et al.(2010), 75% of antibiotic fed to the aquatic animals ended up excreted inwater.

He also reported that level of antibiotics are varies due to differentpractices in every country however Defoird et al. (2011) had estimate theamount of antibiotic in shrimp production in Thailand and Vietnam where bothcountries showed a large variation in usage of antibiotic (as cited in Romero,Feijoó & Navarrete, 2012).  2.4.

2    Antibiotic resistanceAccording to Center for Disease Control andPrevention (2017), antibiotic resistance refers to bacteria that unaffected bythe effect of antibiotics. It occurs when the bacteria evolve in a way toreduce the effectiveness of the antimicrobial agents. Antibiotic resistance hasbeen world’s most pressing public health problems because it can be spreadeasily through the consumption of food that has been contaminated withantibiotic resistance bacteria. Aquaculture animals are the most affected byantibiotic resistance bacteria due to wide usage of antibiotic in farming whichmay be excreted in faeces or by the residual antibiotic that is not consumed bythe aquatic animals. Chakravarty et al.

(2015)found that E. coli in shrimps’ samplefrom Visakhapatnam Fishing Harbour showed 100% resistance to penicillin andtetracycline 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 wereresistant to ciprofloxacin and nalidixic acid. They also found that S. aureus isolates were resistant to aminoglycosides, ?-lactams and tetracycline.

Onthe other hand, Macedo et al. (2016) detected antibiotic resistance S. aureus in fresh marine shrimp whichindicates that shrimps may be a vehicle to spread antibiotic resistant S.

aureus.Due to steady occurrence ofantibiotic resistant bacteria, it is crucial to identify antibiotic resistantbacteria as it will give a major impact towards treatment of foodborne illnessand at the same time contribute a better control of antimicrobial resistance.Monitoring of antibiotic resistant bacteria with accordance to Clinical andLaboratory Standard Institute (CLSI) can help in providing the informationneeded for treatment of ill patients (CLSI, 2015).