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ISOLATION, IDENTIFICATION AND ANTIBIOTIC RESISTANCE

OF ENTEROBACTERICEAE FROM SHRIMP PONDS

IN KUALA BARAM, MIRI, SARAWAK

Arweena Binti Iskandar

RM
267
A796

2014

Master of Environmental Science


(Land Use and Water Resource Management)

2014

usat Kh'dmlt Ma1dumlt Akldemik


!

VE S

MALAYSIA SARAWAK

Faculty of Resource Science and Technology

ISOLA TION, IDENTIFICATION AND ANTIBIOTIC RESISTANCE OF

ENTEROBACTERICEAE FROM SHRIMP PONDS IN KUALA BARAM, MIRI,

SARAWAK

Arweena binti Iskandar (13030160)

Master of Environmental Science

(Land Use and Water Resource Management)

2014

ACKNOWLEDGEMENT

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 ofthis
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.

ii

3.0

Materials and Method ... .. . .. . .. ... . .. ....... ...... .. . ... ........... . ... ..... .......... .. .

3.1 Study Area....................................... .......... .. ..... .. .. ... ... .... .. .. ...


J

4.0

"

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3.2 Collection of Samples... .. . .. . .. . ... ... .. . ... .. . ... .. . ... .. . ... ... ... .. . ... ... ... ... ..

18

3.3 Sample Processing ..... .... .. . ......... ... ... .. . ... ... .. . .. .. ... .... . .. . ......... .. .. ..

18

3.4 Preparation of Eosin Methylene Blue (EMB) Agar.. ... . .. .... ..... ...... ...... ..

19

3.5 Bacteria Isolation from Water Samples......... .. . .. .... ...... .. .... ... ... ... .. ... .

19

3.6 Bacteria Isolation from Shrimp Samples ... .. . ... ... ... ... ... ... .. . .. . ... ... .. . .. . .

20

3.7 Polymerase Chain Reaction Analysis (PCR) ... ... ... ... ... ... ... .. . ... ... .. . ... ...

21

3.8 Identification ofbacteria by Anaytical Profiile Inex (API) 20E Test Kit ......

21

3.9 Preparation of Mueller-Hinton Agar (MHA) .. . .. ... . ... .. . .. . .. ... ... .. .... . .. ....

24

3.10 Antibiotic Susceptibility and Resistance Test........ ... . .. ......... . .. . .. . .....

24

Results ........ . .. .... ... ........... .. .. .. ...... . ... ..... .. ..................... .. .... ... .... .

4.1 Sample Processing .... .. .. , ... '" ........... . .. . .. . ' " .. . ... ... .. . ... ... ... ... ... .... ..

25

4.2 Isolation of Bacteria from Water and Shrimp Samples ..... .. .. .. . .... .. .. . .... ..

28

4.3 Characterization ofbacteria by Polymerase Chain Reaction (PCR) ... ... ..... . ..

29

4.4 Identification ofbacteria by Anaytical Profiile Inex (API) 20E Test Kit... ...

32

4.5 Antibiotic Resistance Test... .. . .. . ... .. . .. ... ... . ........ . .. . .................. .. .....

32

5.0

Discussions.. . ... .. . .. .......... ...... .. .......... .. ... . .. . .. . ..... . ........ ....... .........

34

6.0

Conclusion and Recommendations ... .. . .. . ... ... ... ... .. . .. . .. . ... ... .. . ... ... .. . ... ...

39

7.0

References ..... . ... ... ... .. , ..... .. ... .. .. .... .. . .. .... : ....... . .. . .. .... .. ...... . .. . ... ....
\

Appendixes

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v

40

LIST OF ABBREVIATIONS

Percentage

Degree celcius

Ilg

Microgram

Ml

Microliter

ADH

Arginine dihydrolase

AMP

Ampicillin

API

Analytical Profile Index

AST / ART

Antimicrobial resistance test

Chloramphenicol

CFU

Colony Fonned per Unit

CIT

Citrate

CRO

Ceftriaxone

DNA

Deoxyribonucleic acid

E. Coli

Escherichia coli

EMB

Eosin Methylene Blue

EMBA

Eosin Methylene Blue Agar

gm

Gram

GLU

glucose

H2S

Hydrogen sulphide

IND

Indole

LDC

Lysine decarboxylase

MHA

Mueller-Hinton agar

ml

Millilitre
I

NA Slant

Nutrient Agar Slant

ODC

Ornithine decarboxylase

ONPG

o-nitrophenyl-~- D-galactopyranoside

PCR

Polymerase Chain Reaction

Spp.

Species

TE

Tetracycline

TDA

Tryptophan deaminase

TSA

Tryptic soy agar

URE

Urease

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vi

LIST OF TABLES

Table I

Antibiotics used and its content concentration

Table 2

Total bacteria count for first sampling of water and shrimp

samples cultivation
Table 3

Total bacteria count for second sampling of water and shrimp


samples cultivation

Table 4

Antibiotic susceptibility testing .

Table 5

Antibiotic resistance patterns and multiple drug resistance.

,.

vii

LIST OF FIGURES

Figure I

The chemical structure of ampicillin

Figure 2

The chemical structure of chloramphenicol

Figure 3

The chemical structure of ceftriaxone

Figure 4

The chemical structure of tetracycline

Figure 5

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


study, only spread plate method is applied

Figure 6

Multiple techniques of streaking. In this study, method "D" is


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

Figure 7

of all twenty compartments


Step 2 - Colour comparison test to detennine the outcome of

Figure S

either positive or negative


Figure 9

Step 3 - Key-in the input into the software

Figure 10

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


database

Figure 11

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

Figure 12

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

Figure 13

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


sampling.

Figure 14

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


sampling

Figure 15

Figure 16

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

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

Figure 17

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

viii

Figure 18

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


Figure 19

"

The result for API 20E test kit. Note the changes of colors
which indicates the +ve or -ve reaction

Figure 20 (A - B)

The PCR band for the samples

Figure 21

Dendogram generated from the results of PCR bands

Figure 22

Antibiotic test result - zone of inhibition measurement

ix

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

ABSTRACT
{ntibiotics have been widely used to control bacterial pathogens of aquaculture products.
:k~uaculture is 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.
Kata kunci: Bakteria. ubat-ubatan anti-mikrob. ujian kerentanan. pengasingan. persekitaran
akuakultur

1.0 INTRODUCTION

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 in aqua environment is often linked to water


temperature. Several studies indicated that low temperature increase the ability of E.
2

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


3

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).

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4

PuSlt Khldmat Maklumat Akademik

UNiVERSm MALAYSJA SARAWAK

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:


To isolate and identify Enterobactericeae from the aquaculture environment;

1.
ll.

To determine the antibiotic resistance of Enterobactericeae isolated from the


aquaculture environment.

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5

2.0 LITERATURE REVIEW

.J

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).

2.2.1

Enterobacteriaceae
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

,
6

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
7

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).

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).
8

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).

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9

2.2.5 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,
1999).

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,
1999).

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
10

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.

11

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,

Flavobacterium psychrophilllm, Pseudomonas fluorescens,

V.

ordalii,

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.,
2011).

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
12

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

,
13

antibiotics are used which are:

ampicillin, chloramphenicol, ceftriaxone and

tetracycline.

2.6.1

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).

14

2.6.2

Chloramphenicol

Figure 2: The chemical structure ofchloramphenicol

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,
2006).

2.6.3

Ceftriaxone

Figure 3: The chemical structure ofceflriaxone

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%

,
15

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).

2.6.4

Tetracycline

OH

OH

Figure 4: The chemical-structure oftetracycline

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


usefulness has been reduced with the onset of bacterial resistance (Jones et
al.. 2006). It is named for their four (tetra-) hydrocarbons ring (-cycl-)
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).

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
16