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Arweena Binti Iskandar





Master of Environmental Science

(Land Use and Water Resource Management)


¥usat Kh'dmlt Ma1dumlt Akldemik

! VE



sat Kh'dmlt Ma1dumlt Akldemik ! VE S MALAYSIA SARAWAK Faculty of Resource Science and Technology ISOLATION,

Faculty of Resource Science and Technology


Arweena binti Iskandar (13030160)


Master of Environmental Science (Land Use and Water Resource Management)



ACKNOWLEDGEMENT In the Name of Allah, the Beneficent, the Merciful I take this opportunity to express

In the Name of Allah, the Beneficent, the Merciful

I take this opportunity to express my profound gratitude and deep regards to my supervisor,

Dr Samuel Lihan, Senior Lecturer, Department of Molecular Biology Faculty of Resource Science and Technology, UNIMAS, for his exemplary guidance, monitoring and constant encouragement throughout the course of this thesis. The blessing, help and guidance given

by him time to time is very precious .

I also take this opportunity to express a deep sense of gratitude to my family and to my and

Dr Tay Meng Guan, Coordinator for SLUSE programme their cordial support, valuable information and guidance, which helped me in completing this task through various stages. Also for their patience, motivation, and encouragement given towards the completion of this project. Without them~ I would not be able to c~mplete this project on time.

I wish to acknowledge the help/facilities provided by Faculty of Resource Science and

Technology (FRST) (particularly virology lab). labatan Kesihatan Bahagian Miri and the shrimp farming's owner in Kuala Bararn for the cooperation, equipment used throughout this project duration and importantly, and consenting this study to be carried out. Without their permission and cooperation, this study would not be possible.

My completion of this,project could not have been accomplished without the support of two lovely students: Miss Rynie and Miss Waynie Valeria whom assists me during my absenteeism in the laboratory (while I was in Miri). Your hard works and patience amazed me and it will take more than words can say to reward your assistance.

Last but not least, thank you to all my fellow classmates and all my friends for your moral support and for being a good friend to me.

Thank you.




Materials and Method


3. 1 Study Area




3.2 Collection of Samples



3.3 Sample Processing


3.4 Preparation of Eosin Methylene Blue (EMB) Agar



3.5 Bacteria Isolation from Water Samples



3.6 Bacteria

Isolation from Shrimp Samples





3.7 Polymerase Chain Reaction

Analysis (PCR)



3.8 Identification of bacteria by Anaytical Profiile Inex (API) 20E® Test Kit



3.9 Preparation of Mueller-Hinton Agar (MHA)




Antibiotic Susceptibility and Resistance Test







' "


4.1 Sample Processing

4.2 Isolation of Bacteria from Water and Shrimp Samples





4.3 Characterization of bacteria

by Polymerase Chain Reaction (PCR)


4.4 Identification of bacteria by Anaytical Profiile Inex (API) 20E® Test Kit



4.5 Antibiotic Resistance Test







Conclusion and






















Degree celcius






Arginine dihydrolase




Analytical Profile Index


Antimicrobial resistance test




Colony Fonned per Unit






Deoxyribonucleic acid

E. Coli

Escherichia coli


Eosin Methylene Blue


Eosin Methylene Blue Agar






Hydrogen sulphide




Lysine decarboxylase


Mueller-Hinton agar





NA Slant

Nutrient Agar Slant


Ornithine decarboxylase




Polymerase Chain Reaction






Tryptophan deaminase


Tryptic soy agar





Spp. Species TE Tetracycline TDA Tryptophan deaminase TSA Tryptic soy agar URE Urease , vi


Table I

Antibiotics used and its content concentration I

Table 2

Total bacteria count for first sampling of water and shrimp

Table 3

samples cultivation Total bacteria count for second sampling of water and shrimp

Table 4

samples cultivation Antibiotic susceptibility testing .

Table 5

Antibiotic resistance patterns and multiple drug resistance.






Figure I

The chemical structure of ampicillin



The chemical structure of chloramphenicol

Figure 3

The chemical structure of ceftriaxone



The chemical structure of tetracycline

Figure 5

Illustration of the pour / spread plate method. In the current

Figure 6

study, only spread plate method is applied Multiple techniques of streaking. In this study, method "D" is

Figure 7

used while streaking Step I - Dispense saline suspension of organisms into cupules

Figure S

of all twenty compartments Step 2 - Colour comparison test to detennine the outcome of either positive or negative



Step 3 - Key-in the input into the software

Figure 10

Step 4 - the identification data will be provided by the online database




count (cfu/ml) for water sample during first sampling

Figure 12


Bacterial count (cfu/ml) for shrimp sample during first sampling

Figure 13

Figure 14

Figure 15

Bacterial count (cfu/ml) for water sample during second

sampling. Bacterial count (cfu/ml) for shrimp sample during second

sampling I The growth of E.coli where greenish colour is visible on EMB


Figure 16


Agar The growth of E.coli where greenish colour is visible on EMB

Figure 17

Agar The growth of unidentified bacteria where red colour (as seen in Fig.IS) and grey colour (as in Fig. 19) is visible on EMB Agar after incubation for 24 hours


Figure 18

Figure 19

Figure 20 (A - B)

The growth of unidentified bacteria where red colour (as seen in Fig.I8) and grey colour (as in Fig. 19) is visible on EMB Agar after incubation for 24 hours The result for API 20E® test kit. Note the changes of colors which indicates the +ve or -ve reaction The PCR band for the samples





Dendogram generated from

the results of PCR bands



Antibiotic test result - zone

of inhibition measurement



Isolation, Identification and Antibiotic Resistance of Enterobactericeae from Shrimp Ponds in Kuala Baram, Miri, Sarawak

Arweena binti Iskandar

Master of En vironmental Science Faculty of Science and Technology Universiti Malaysia Sarawak


{ntibiotics have been widely used to




pathogens of aquaculture products.

:k~uacultureis becoming more economically important to increase local fish and shrimp production for food security. Thus, antimicrobial drugs are therefore used to tackle the disease problems and in some cases, they are used as growth promoter. However, prolong usage of the drugs may I ad to severe health problems to human as well as the resistance problem among the bacteria present The present study aims to detdermine the resistancy of the Enterobacteriaceae isolated from water and shrimp from the aquaculture environment. A total of 20 water samples and 40 shrimps were collected from 10 aquaculture pond in a farm in Kuala Baram, Miri, Sarawak. Isolates were first screened by using peR, followed by identification with API 20E Test Kit, and finally conducting the antimicrobial drugs resistant test. Four antimicrobial drugs were used in this study: ampicillin (AMP), chloramphenicol (C), ceftriaxone (eRO), and tetracycline (TE). The results reflected that the 31 bacterial isolates were classified into 4 different families: Enterobacteriaceae. Brucellaceae. Pseudomonadaceae; and Xanthomonadaceae. Antimicrobial resistance test rerevealed that 93.5% of the isolates were resistant to AMP, while others were susceptible to e, eRO and TE. This study revealed the presence of multiple antibiotic resistant bacteria from aquaculture environment.

Key words: bacteria, antimicrobial drugs, resistancy test, isolates, aquaculture environment

ABSTRAK Antibiotik telah digunakan secara meluas untuk mengawal patogen bakteria di dalam produk akuakultur. Sektor akuakultur menjadi salah satu sumber ekonomi yang penting sebagai satu cara meningkatkan pengeluaran ikan dan udang tempatan untuk tujuanjaminan bekalan makanan yang mampan. Oleh yang demikian, ubat-ubatan antimikrobial digunakan untuk menangani masalah penyakit. dan dalam sesetengah kes. ia digunakan untuk menggalakkan pertumbuhan produk ternakan. Walau bagaimanapun, penggunaan ubat-ubatan antimikrobial yang berpanjangan boleh membawa kepada masalah kesihatan yang teruk dan juga masalah kerintangan bakteria. Oleh itu. projek ini mengkaji kerentangan Enterobacteriaceae yang diasingkan daripada air dan udang dari persekitaran akuakultur. , Sebanyak 20 sampel air dan 40 sampel udang telah diambil dari 10 buah kolam ternakan udang di sebuah ladang di Kuala Baram. Miri. Sarawak. Sampel yang diasingkan terlebih dahulu dikenal pasti dengan menggunakan peR. diikuti dengan 20E API Test Kit dan seterusnya diuji untuk kerentanan terhadap ubat-ubatan anti-mikrob berikut: ampicillin (AMP). chloramphenicol (e). ceftriaxone (eRO). and tetracycline (TE). Keputusan menunjukkan bahawa 31 sampel yang diasingkan telah dikelaskan kepada 4 keluarga yang berbeza iaitu:

Enterobacteriaceae. Brucellaceae. Pseudomonadaceae; dan Xanthomonadaceae. Ujian kerentanan terhadap uabt-ubatan antimikrobial menunjukkan bahwa 93.5 peratus daripada sampel yang diasingkan tahanlrentan kepada AMP. manakala yang lain adalah lebih sensittf kepada C. eRO dan TE. Kajian ini mendedahkan kehadiran kerentanan terhadap antimikrobial dari persekitaran akuakultur.













1.0 INTRODUCTION Shrimp aquaculture has become a commercially important industry and is widespread throughout tropical


Shrimp aquaculture has become a commercially important industry and is widespread throughout tropical countries . Bacterial disease has been cited as the single largest source of economic loss in the aquaculture industry (Russo et aI., 2012; Erkmen, 2004). The distribution of Vibriosis is worldwide, caused by various Vibrio spp. and they are responsible for the high mortality and severe economic loss in shrimp industry in all the shrimp producing countries. Various diseases have been studied for many years and reported to cause not only serious infections and also lower shrimp production (mortality, tissue lesion or necrosis, body malformation, low growth). Problems with the bacterial disease and the estimated loss of wild stocks are unknown, except for a few reports on the occurrence of bacteria in the processed iced storage shrimp (Adedeji et aI., 2012; Hossain et al., 2012).

Bacteria, present in aquaculture settings may be transmitted to humans who come in contact with this ecosystem. For instance, the cholera epidemic in Ecuador began among persons working on shrimp fanns. Multidrug-resistance was' present in non­ cholera Vibrio infections that were pathogenic to the shrimp. Bacteria from the aquaculture ecosystem may also be transmitted directly to humans through handling of fish and shrimp (Hossain et al., 2012). Fish pathogen Streptococcus iniae, for example, has caused invasive infections in persons who handled aquacultured tilapia; and also Streptococcus iniae was isolated from the aquaculture ecosystem and from purchased fish in grocery stores (Adedeji et aI., 2012). Bacteria on fish and shrimp may also be transmitted to humans when the aquacultured fish and shrimp are eaten, or when other foods, which ha 'e been cross-contaminated by ba~teria from fish or shrimp, are eaten (Heuer et al., 2009). For example, in Japan infections have been linked to the consumption of aquacultured fin fish. Furthermore, Salmonella spp., a common cause of foodbome disease, has been isolated from aquacultured fish and shrimp ponds. These and other reports indicate that bacteria present in aquaculture ecosystems can be transmitted to humans (Adedeji et aI., 2012; Adedeji et al., 2011).

Bacteria' s persistency and survival

temperature . Several studies indicated that low temperature increase the ability of E.

in aqua environment is often linked to



coli to survive in a variety of aquatic condition (Lukas et al., 2013). The perception of people are low temperature would be unsuitable for bacterial survival. In addition to the effect of temperature on survival, recent findings suggest that sediments may be reservoir for faecal bacteria in aqua environment. Although bacteria causing diseases in penaeid shrimp are considered opportunistic and manageable, the economic implication of losses due to mortality of shrimp or rejection of the infected shrimp is enormous (Mohammad et al., 2008; Victoria et al., 2006). Antimicrobial drugs have been widely used to tackle the disease issue. Apart from controlling diseases, antibiotics are used as growth promoter which aims in enhancing the meat production and nutritional value. However, prolong usage of the drugs may lead to severe health problems (Srinivasan et al., 2009). Some microorganisms are naturally resistant to certain antibiotics and there are several reasons why these microorganisms may have an inherent natural resistance to an antibiotic. Some of the reason are: lack of antibiotic-inhibited structure, reduced permeability, alteration and subsequent inactivation of the antibiotic, target modification, developing a resistant biochemical pathway by genetic change or physiological switch, or efflux of the antibiotic (Tendencia et al., 2001).

In Malaysia, the aquaculture activity has begun in the 1920's with extensive polyculture in ex-mining pools of introduced Chinese carps, mainly the bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix) and grass carp (Ctenopharyngodon idellus). In the mid 1930's, marine shrimp trapping ponds were first developed in lohore, the southern state of Peninsular Malaysia. In the early 1940's, the culture of blood cockles (Anadara granosa) began. This was followed in the mid 1950's by the extensive culture of freshwater fish in earthen ponds (Ali, 2007).


In the early 1970's, great changes in aquaculture began to take place, when the semi- intensive culture of shrimp was developed in lohore. Shrimp culture was characterized by very low stocking density combined with pond fertilization. During the same period, floating net cage culture of marine fish, mainly the green grouper (£pinephelus coioides), began to take place, followed by the raft culture of green mussels (Thalathiah et al., 1993; Tan, 1994). By the early 1990's, aquaculture activities were further enhanced with the introduction of intensive commercial aquaculture with very high stocking density and complete dependence on supplementary feeding. Commercial aquaculture was made possible through the establishment of government


and privately owned fish and shrimp hatcheries, which started in the 1980's. The setting up of private feed mills in the 1980's also contributed to the commercialization of aquaculture (Thalathiah et al., 1993; Tan, 1994; Ali, 2007).


Since the year 1920's, aquaculture activity in Malaysia has developed quickly and is now an important activity. Several culture practices are used. Brackish water aquaculture is the predominant practice, but there is also freshwater pond aquaculture and marine aquaculture. A wide variety of species is cultured, including shellfish, freshwater species and marine finfish. Aquaculture is becoming important as a way of increasing local production for food security and increasing export revenues. The sector has become a priority area in the government's most recent policy programme for 1998-2010. The aim by 2010 is to increase aquaculture production by 200 percent (Fisheries and Aquaculture Department, 2014). However, difficulties in land acquisition, rising production costs, lack of skilled labour and threat of diseases are obstacles which impede the development of aquaculture. Research is being given priority due to the importance of aquaculture as an alternative source of fish supply.

Aquaculture is becoming economically more important as a way of increasing local fish production for food security. Aquaculture production is still very small compared to capture fisheries: it contributes less than 0.2 percent to GOP. However, it has succeeded in producing high value species for the domestic market as well as for the export market. This aspect is capitalized on in the poverty alleviation programme involving the very poor to improve their household income. The culture of bivalve molluscs is the easiest with few overheads and has proved very successful among the poor households in coastal areas. Seaweed culture in Sabah has proved to be very successful in improving the livelihood of the poor fishennen because the produce is sold in a dried fonn and exported to the Philippines. In the inland area, the fanning of tilapia in earthen ponds through government - assisted projects has been very successful among the poor households because it breeds naturally. Culture in floating net-cages in lakes and reservoirs through government-assisted projects, has also proved to be successful and likely to improve livelihoods, as well as providing a cheap source of protein (Fisheries and Aquaculture Department, 2014).




PuSlt Khldmat Maklu mat Akademik


The main fisheries authority at federal level is the Ministry of Agriculture and Agro­ based Industry (MOA). With regard to aquaculture, the Director-General of Fisheries, head of the Fisheries Department, is vested with orientation powers for the development of marine and inland farming, in consultation with the concerned State Authority. In particular, the promotion of inland aquaculture may involve the creation of experimental aquaculture stations for demonstrative purposes, fish-breeding facilities and training centres. An important enforcer in the development of the national maritime policy is the Maritime Institute of Malaysia (MIMA), a policy research institute set-up by the Malaysian Government to specifically deal with national, regional and global maritime issues. The Freshwater Fisheries Research Centre operates within the Fisheries Department of the Ministry of Agriculture, for the development of freshwater aquaculture, and the conservation and management of aquatic resources (Fisheries and Aquaculture Department, 2014).

In view of increasing concerns surrounding the evolution of antibiotic resistant bacteria in aquaculture environments, the present study was undertaken, with the main objective: to determine the antibiotic resistance expressed by Enterobactericeae isolated from water and shrimp cultured "at aquaculture pond at Kuala Baram, Miri, Sarawak.

The specific objectives of this study are:

1. To isolate and identify Enterobactericeae from the aquaculture environment; To determine the antibiotic resistance of Enterobactericeae isolated from the aquaculture environment.






2.1 The definition of aquaculture


The FAO defines aquaculture as fanning of aquatic organisms including fish, molluscs, crustaceans and aquatic plants. Farming implies some sort of intervention in the rearing

process to enhance production, such as regular stocking, feeding, and protection from predators. Fanning is also implied as the ownership of an individual or corporate of the stock being cultivated. For statistical purposes, aquatic organisms which are harvested by an individual or corporate body which has owned them throughout their rearing period contribute to aquaculture while aquatic organisms which are exploitable by the public as a common property resource, with or without appropriate licences, are the harvest of fisheries (FAO, 1997)

2.2 Bacteria associated with aquaculture practices The hazards associated with human pathogenic bacteria in the finfish and crustaceans produced in aquaculture can be divided into two groups, bacteria naturally present in the aquatic environment (indigenous bacteria) and bacteria being introduced into the aquatic environment or contaminated by human or animal faeces. Hazards may also arise through the introduction of bacteria during post-harvest handling and processing (Cabral et aI., 20 10; Buras, 1993).



Pathogenic enterobacteria can be introduced into aquaculture ponds by animal (including birds) manure and human waste. Study shows that there is evidence of the rapid die-off of enteric organisms ru;td viruses in well-managed ponds (Edwards et al., 1990). However, there are significant numbers of organisms can be found in products harvested from waste-fed systems, and such products therefore pose a potential health risk (Buras, 1993).

Salmonella spp. is one of the important causes of human gastrointestinal diseases and many seafood-importing countries will not accept products containing this pathogen. Studies have indicated that there is a higher prevalence of Salmonella in tropical than in temperate waters and they may be naturally



present in some tropical aquatic environments. It is well established that aquatic birds spread these organisms and other pathogens in the environment (Fenlon, 1983; Beveridge, 1995). Salmonella spp. have been reported in fish ponds:

surveys have shown that 21% of Japanese eel culture ponds, 5% of North American catfish ponds, and 22% of shrimp ponds in one of the major shrimp­ exporting countries in south-east Asia (Reilly et aI. , 1992) are contaminated with the organisms. Salmonella spp. have been isolated from tropical aquaculture systems where faecal wastes are not used as fertilizer; this is most likely due to unavoidable contamination by scavenging birds and other animals. While Salmonella spp. tend to be associated with the intestinal tracts of warm-blooded animals, they have also been detected in the gut of tilapia and carp grown in waste-fed and non-waste-fed aquaculture ponds (Buras, 1993; Iyer et al., 2009).

Escherichia coli is a common inhabitant of intestinal tract of humans and animals, and can be easily disseminated in different ecosystems through the food chain and water. Animal food products are an important source of E. coli as faecal contamination of carcasses at the slaughterhouse is frequent (Clark, 2007). These microorganisms and th~ir possible resistance determinants may be transmitted to humans if these foods are improperly cooked or otherwise mishandled. The level of antibiotic resistance in E. coli represents a useful indicator of the resistance dissemination in bacterial populations. There are some reports in which antibiotic susceptibility of E. coli isolates from healthy humans or animals have been studied, but in few cases comparative results have been shown or isolates from foods analysed (Garcia et aI., 2010). E. coli has been shown to exchange genetic material with other bacterial species and it is possible that this organism may pass antibiotic resistance genes to transient bacterial pathogens that cause disease in humans (Fratamico et al., 2006). A study done by Jaime et al. in 2012 noted that in recent years, accumulating problems with resistant bacteria, leading to predictions that we are back the period before the discovery of antibiotics. One of way around this problem is to introduce new antibacterial preparation which could be isolate from some wild medical plants with antimicrobial effects or it could be operates on a locking mechanism of virulence, more precisely, a type III (T3SS) secretion system. Infections caused by resistant strains of microorganisms causing costly treatment of animals and


humans. Such infections prolong the pathological condition and if not treated with the right antibiotics may be increased mortality (Anonymous, WHO, 1997).

A number of other human pathogenic enterobacteria, including Shigella spp.

have occasionally been isolated in aquaculture systems and products (Alderman et al., 1998). However, on the basis of epidemiological evidence, there appears

to be very little risk of infection associated with the consumption of farmed fish

products (Anonymous, WHO, 1997).

Campylobacter spp. are very common and impOltant causes of diarrhoeal illness

in humans. They are commonly found in the gut of warm-blooded animals,

especially poultry, but they are not part of the normal flora of unpolluted aquatic

environments. However, these organisms are frequently isolated from wastewater (Ryan et al., 2004). There is very little information on the occurrence

of Campylobacter spp. in aquaculture, although the use of poultry manure for

fertilizing ponds may constitute a public health risk in inland and coastal aquaculture environments. There are some reports of the occurrence of Campylobacter spp. in bivalves, but there is insufficient information about their occurrence in finfish and crustaceans. Available data suggest that the risk of Campylobacter infection associated with the consumption of farmed fish

products is low (Anonymous, 1997).

of farmed fish products is low (Anonymous, 1997). 2.2.2 Vibrio spp. Vibrios are generally salt-tolerant

2.2.2 Vibrio spp.

Vibrios are generally salt-tolerant organisms occurring naturally in marine and brackish-water environments in both tropical and temper ate regions, although Vibrio cholerae and V. mimicus also occur in fresh water. Vibrios have also been isolated from sediments, plankton ton, molluscs, finfish and crustaceans (Hossain et al., 2012). While several studies have shown that the occurrence of vibrios does not correlate with numbers of faecal coliforms, there is a positive correlation between their occurrence and the admixture in water of contaminated human waste. There is also a positive correlation between water temperature and both the number of human pathogenic vibrios isolated and the number of reported infections. A seasonal correlation is particularly marked for V. vlIlnificus and V. parahaemolyticlls (Hossain et al., 2012; Anonymous, 1999).


Currently, 12 species of Vibrio are known to be associated with human infections acquired by consumption of contaminated foods and water. Some human pathogenic Vibrio spp. may also be fish pathogens (Adeleye et al., 2008). In general, the infectious dose necessary to cause intestinal disease is high, and the risk associated with eating fish is therefore likely to be low. For example, about a million organisms must be ingested to cause cholera. Although more than 150 serotypes have been identified, only V. cholerae Oland 0139 cause cholera; non-O 1 V. cholerae can cause diarrhoea, abdominal cramps, nausea and fever (Bhaskar et al., 1994). While cholera has been associated with the consumption of raw fishery products, there are no reported cases resulting from the consumption of commercially imported farmed finfish and crustaceans (Hossain et ai., 2012).

2.2.3 Aeromonas spp. and Plesiomonas spp.

Aeromonas and Plesiomonas spp. constitute part of the normal aquatic bacterial flora, with Plesiomonas spp. occurring more common in tropical waters. A. hydrophila is the species most often associated with food borne disease (Morgan et al., 2008) and P. shigelloides has been implicated in outbreaks of gastroenteritis from the consumption of fish (Anonymous, 1999; Bhat et al., 1994). Epidemiological evidence suggests public health risks from Aeromonas and Plesiomonas spp. in farmed fish are low.

2.2.4 Clostridium botulinum

Clostridium botulinum is a ubiquitous, spore-forming, anaerobic organism that produces a neurotoxin causing life-threatening fQodborne illness. This organism can be grouped into seven types on the basis of the antigenic nature of the neurotoxin produced. C. botulinum type E is naturally found in aquatic environments and is often isolated from fish. However, if the fish are properly handled and processed to prevent growth of the organism and production of the toxin there should be no risk of botulism (Anonymous, WHO, 1999).



prevent growth of the organism and production of the toxin there should be no risk of


Listeria monocytogenes

Although Listeria monocytogenes IS a foodborne pathogen, fanned finfish and crustaceans have not been implicated in any epidemic outbreaks. However, seafood, including fish, smoked fish, smoked mussels and smoked salmon, has caused sporadic cases of listeriosis in vulnerable populations. L. monocytogenes is frequently isolated from aquaculture products from temperate regions but is rarely reported in tropical fishery products. Fish produced in temperate inland aquaculture systems may be contaminated with L. monocytogenes and thus present a potential health risk when consumed raw or without heat treatment (Anonymous, WHO,


2.2.6 Other bacteria

A number of other bacteria pathogenic to humans, including Erysipelothrix

rhusiopathiae, Leptospira interrogans, Yersinia enterocolitica, Pseudomonas spp.,

Streptococcus iniae and Mycobacterium spp. are widely disseminated in nature, including the aquatic environment. There is little evidence, except in the case of M. marinllm, that their distribution is affected by aquaculture activities. M. marinllm has been isolated from disease outbreaks in food fish and ornamental fish. There are

no reported cases of illness caused by M. marinum associated with the consumption of farmed finfish or crustaceans. On the other hand, M. marinum and S. iniae may present occupational hazards to workers handling infected fish (Anonymous, WHO,


2.3 The use of antimicrobial in aquaculture

Antibiotics and probiotics have been widely used to control bacterial pathogens of shrimp (Hossain et al., 2012). In hatcheries, it is common practice to use anti biotics for prophylaxis and chemotherapy, especially when larval development is hampered. However, administration of antibiotics to infected stocks of shrimp is usually impracticable in large scale culture enterprises, as the only routes of administration are through the culture water or in pelletized feed (Paul, 2008). Antibiotic resistant strains have caused mass mortality in cultured P. monodon larvae and Macrobrachillm rosenbergi;. Throughout Asia, shrimp farmers use antibiotics in large quantities . Potential consequences of antibiotics use in culture and in animal feeds are the transfer

of resistant characters to bacteria thereby leading to the development of antibiotic


resistant bacterial strains, and thereby reduced efficacy of antibiotic treatment for human and animal diseases (Felipe et al., 2013; lawahar et al., 201l). Resistance of marine fish and shellfish pathogens to commonly used antibiotics has also been reported in lapan and Denmark. Antibiotic resistance of isolates of Vibrio spp. from aquaculture systems of fish, shellfish and sea foods has been found in Saudi Arabia (Felipe et al., 2013). The luminescent species infecting shrimp do not always respond to antibiotics and other chemical control methods, numerous efforts have been made to find new chemotherapeutic agents to replace those antibiotics against which the disease causing bacteria have become resistant (Paul, 2008; Samuel et al., 20ll).

Because antimicrobial agents used in aquaculture are administered by mixing them with feed which is dispersed in the water, use of antimicrobial agents in aquaculture directly doses the environment, which results in selective pressures in the exposed ecosystem (Felipe et al., 2013; lawahar et aI., 20ll). In several countries A. salmonicida is frequently resistant to multiple drugs including sulphonamides, tetracycline, amoxicillin and quinolones, antimicrobial agents which are commonly used in aquaculture. Similar correlations between antimicrobial agents used in aquaculture and antimicrobial resistanc~ are also reported among other aquaculture pathogens (Lukas et al., 2013; Srinivasan et aI., 2008).

2.4 Mechanism of antibiotic resistance

Antibiotics are generally designed to target chromosomally encoded proteins of single bacterial species which is responsible for causing anomaly in living system (Felipe et al., 2013; Adedeji et al., 2011; lawahar et al., 201l). A mutation in bacterial cell can develop antibiotic resistance to that targeted protein, which also renders the original protein to change its form and normal function. When no antibiotics are present in the environment, these resistant bacteria will be at a disadvantage and therefore will not spread or exist any longer. So, single chromosomal mutations alone are not the sole cause of bacterial resistance in the environment.


2.5 Effects of aquacultural use of antimicrobials on animal and human health The most obvious

2.5 Effects of aquacultural use of antimicrobials on animal and human health The most obvious detrimental effect of extensive use of antimicrobials in aquaculture is selection of fish and shrimp pathogens resistant to multiple antimicrobials which in turn produce difficult or impossible to treat epizootics (Felipe et al., 2013; Renata et al., 2012). The clinical problems generated in veterinary and human medicine by antimicrobial-resistant bacteria are well reviewed, and fish and shrimp pathogens resistant to multiple antimicrobials used in aquaculture have been described. These include Aeromonas salmonicida, A. hydrophila, A. caviae, A. sobria, E. ictaluri, E. tarda, P. damselae piscicida, V. anguillarum, V. salmonicida, V. ordalii, Flavobacterium psychrophilllm, Pseudomonas fluorescens, Streptococcus iniae, Renibacterillm salmonicarum, Yersinia ruckeri and Piscirickettsia salmonis (Felipe et aI., 2013; Renata eta/., 2012).

Some of these pathogens, e.g. Edwarsiella, Aeromonas, and Streptococcus, can infect humans and generate antimicrobial-resistant zoonotic infections. Kluyvera spp. and S. maltophilia are additional aquatic bacteria related to fish that are emerging as opportunistic human pathogens (Felipe et aI., 2013; Renata et al., 2012). Such an occurrence could increase quinolone resistance and increase quinolone concentrations required to prevent chromosomal mutations to these antimicrobials, and as a result complicate treatment of infections caused by these pathogens. While selection of antimicrobial-resistant bacteria in normal intestinal and other flora of fish as a result of aquacultural use of antimicrobials has not been extensively investigated, it is reasonable to suppose that antimicrobial resistance determinants present in normal piscine flora could be the source of resistance (Renata et aI., 2012; lawahar et aI.,



There is strong laboratory and field evidence for readily detectable frequencies of HGT between bacteria in the aquatic environment and human pathogens, as would be expected of genetic exchange communities linked by HGT in spite of the oligotrophy of the aquatic environments. As a result of HGT, these new genetic entities may be incorporated into the pangenome of terrestrial bacteria including human pathogens, linking the aquatic and terrestrial resistomes and complicating the treatment of human infections (Renata et aI., 2012; Paul, 2008). The power of HGT to generate genetic diversity from aquatic bacteria is demonstrated by the ability of human intestinal


Bacteroides to acquire genes needed for degradation of algal polysaccharides from aquatic bacteria. In addition

Bacteroides to acquire genes needed for degradation of algal polysaccharides from aquatic bacteria. In addition to selection and dissemination of antimicrobial-resistant bacteria, excessive use of antimicrobials in aquaculture can potentially have other detrimental impacts on human health (Felipe et al., 2013). Fish and shrimp products for human consumption can become contaminated with antimicrobial residues at doses higher than Maximum Residue Limits. When such products are eaten, they can potentially alter the human normal intestinal flora, select for antimicrobial-resistant bacteria, and aid infection with human pathogens while further facilitating HOT of antimicrobial resistance.

Passage of antimicrobials to humans in fish and shrimp meat may be more common than supposed since regulatory agencies frequently detect antimicrobial residues in fish for human consumption despite the low proportion of aquacultured fish and shrimp tested for the presence of these drugs (Caprioli et al., 2000). Ingestion of free­ ranging (wild) fish, shellfish and crustaceans from areas surrounding aquaculture sites can also result in passage of antimicrobials used in aquaculture to the human intestine since antimicrobials can reach other animals near these sites and remain in their tissues for some period of time (Boogard et al., 2000). Similarly, antimicrobial resistant bacteria selected in aquaculture sites can contaminate marketed aquacultural produce. In addition, there is the reasonable possibility that workers in food mills and aquaculture sites will become exposed to antimicrobials and antimicrobial resistant bacteria by aerosols and by direct contact with medicated food in aquacultural areas where annual usages of antimicrobials run into the metric tons, again shifting the normal flora of skin, intestine and mucosa of these workers towards antimicrobial­ resistant bacteria (Boogard et al., 2000; Caprioli et aI., 2000).

2.6 Antibiotics used in the study

Antibiotics are natural compounds. They are produced by a wide range of fungi and bacteria for inhibiting or killing other microorganisms. Many natural antibiotics have been structurally modified in the 1aboratory to enhance their efficacy. These are said to be semisynthetic antibiotics (Cabello, 2006). For the purpose of this current study, four
















tetracycline.   2.6.1 Ampicillin   Figure 1: The chemical structure qf ampicillin Ampicillin,

Figure 1: The chemical structure qf ampicillin

Ampicillin, a ~-lactam antibiotic, is the semisynthetic penicillin (Madigan and Martinko, 2006). It binds to and inhibits a number of enzymes in the bacterial membrane that are involved in the synthesis of the cell wall (Waxman and Strominger, 1983).


The ampicillin resistance (ampr) gene is carried on the plasmid codes for an enzyme that is secreted into the periplasmic space of the bacterium, where it catalyzes hydrolysis of the ~-lactam ring, with concomitant detoxification of the drug (Sykes and Mathew, 1976). In gram-negative bacteria, several lactam resistance genes are located as cassettes in class 1 integrons (S0lUI11 et al., 2002). Normally bla genes in gram-positive bacteria such as staphylococci are transposon located on small transferable or mobilizable plasmids (Yazdankhah et al., 2000; Sidhu et al., 200 l). Besides, the bla genes have been found in staphylococci from humans, dairy cattle and pet animals and have also been found in the environment of a small animal clinic (S0rum et al., 2002).




2.6.2 Chloramphenicol Figure 2: The chemical structure of chloramphenicol Chloramphenicol is an aromatic compound.

Figure 2: The chemical structure of chloramphenicol

Chloramphenicol is an aromatic compound. It binds to the ribosomal 50S subunit and inhibits protein synthesis. Thus it is specific for ribosomes of bacteria (Madigan and Martinko, 2006). The chloramphenicol resistance (cat) gene codes for a tetrameric, cytosolic protein that, in the presence of acetyl coenzyme A, catalyzes the formation of hydroxyl acetoxy derivatives of chloramphenicol that are unable to bind to ribosomes (Dan et ai., 2008). This protein is an R plasmid-encoded enzyme (Madigan and Martinko,





and Martinko,   2006). 2.6.3 Ceftriaxone Figure 3: The chemical structure of ceflriaxone

Figure 3: The chemical structure of ceflriaxone

Ceftriaxone is a white crystalline powder readily soluble in water, sparingly soluble in methanol, and very slightly soluble in ethanol. The pH of a 1%



aqueous solution is approximately 6.7. The syn-configuration of the methoxyimino moiety confers resistance to ~-lactamase enzymes produced by many Gram-negative bacteria. The stability of this configuration results in increased activity of ceftriaxone against otherwise-resistant Gram­ negative bacteria. Ceftriaxone inhibits bacterial cell wall synthesis by means of binding to the penicillin-binding proteins (PBPs). inhibition of PBPs would in turn inhibit the transpeptidation step in peptidoglycan synthesis which is required for bacterial cell walls (S0fum et al., 2002).



OH 0 OH o

Figure 4: The chemical-structure of tetracycline

Tetracyclines are a group of broad-spectru, antibiotics whose general

usefulness has been reduced with the onset of bacterial resistance (Jones et


derivation (-ine). Tetracycline antibiotics are protein synthesis inhibitors, inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA

translation complex (S0rum et aI., 2002). Tetracyclines also have been found to inhibit matrix metalloproteinases. This mechanism does not add to their antibiotic effects, but has led to extensive research on chemically modified tetracyclines or CMTs (like incyclinide) for the treatment of rosacea, acne, diabetes and various types of neoplasms (Jones et aI., 2006).

2006). It is named for their four (tetra-) hydrocarbons ring (-cycl-)

There are three (3) mechanisms on how the cells can become resistant to tetracycline: enzymatic inactivation of tetracycline, efflux, and ribosomal protection. The rarest type of resistance is through, where the drug is