B I O D I V E R S IT A S ISSN: 1412-033X

Volume 19, Number 2, March 2018 E-ISSN: 2085-4722

Pages: 480-488 DOI: 10.13057/biodiv/d190215

Identification of potentially pathogenic bacteria from tilapia

(Oreochromis niloticus) and channel catfish (Clarias batrachus) culture
in Samarinda, East Kalimantan, Indonesia

ESTI HANDAYANI HARDI1,♥, RUDY AGUNG NUGROHO2, GINA SAPTIANI1, RIA SARINAH1,
MAULINA AGRIANDINI1, MIRA MAWARDI3
1
Department of Aquaculture, Faculty of Fisheries and Marine Sciences, Universitas Mulawarman. Jl. Gunung Tabur, Gunung Kelua, Samarinda Ulu,
Samarinda 75123, East Kalimantan, Indonesia. Tel./fax: +62-541-749159, ♥email: [email protected]
2
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Mulawarman. Jl. Barong Tongkok No 4. Gunung Kelua,
Samarinda 75123, East Kalimantan, Indonesia
3
Main Center for Freshwater Aquaculture Fisheries. Ministry of Marine Affairs and Fisheries. Jl. Selabintana No. 37, Sukabumi 43114, West Java,
Indonesia

Manuscript received: 21 August 2017. Revision accepted: 23 February 2018.

Abstract. Hardi EH, Nugroho RA, Saptiani G, Sarinah R, Anggraidini M, Mawardi M. 2018. Identification of potentially pathogenic
bacteria from tilapia (Oreochromis niloticus) and channel catfish (Clarias batrachus) culture in Samarinda, East Kalimantan, Indonesia.
Biodiversitas 19: 480-488. This research was conducted to isolate, identify, and characterize pathogenic bacteria from feces and water of
tilapia (Oreochromis niloticus) and channel catfish (Clarias batrachus) culture from two selected locations in Samarinda, East
Kalimantan, Indonesia. Bacteria were cultured and isolated on NA, TSA, and BHIA media at 30oC for 24 h. Colonies of the isolated
bacteria were characterized morphologically in terms of their shape, chromogenesis, edge, and size. Antibiotic sensitivity test on each
bacterial isolate was completed using inhibition zone tests. Commercial antibiotics used in this research were nitrofurantoin,
ciprofloxacin, oxytetracycline, chloramphenicol, nalidixic acid, gentamicin, and norfloxacin. Koch’s postulates test was done by
intraperitoneal injection of bacterial suspension to tilapia (15 g weight) at 103-109 CFU mL-1 in triplicates to determine the pathogenicity
of each bacterium. Overall, there were 37 isolates obtained from different sources and growth media that belonged to 14 species:
Acinetobacter calcoaceticus (1 isolate), Aerococcus urinae (2 isolates), Aerococcus viridans (1 isolate), Aeromonas hydrophila (1
isolate), Citrobacter freundii (5 isolates), Enterobacter amnigenus (2 isolate), Enterobacter cloacae (4 isolates), Escherichia coli (3
isolates), Listeria sp. (1 isolate), Niseria sp. (4 isolates), Pantoea spp. (1 isolate), Pseudomonas aeruginosa (1 isolate), Staphylococcus
aureus (9 isolates), and Streptococcus iniae (2 isolates). Sixteen of the isolates were grown in BHIA medium, 12 isolates in TSA
medium and 9 isolates in NA medium. The highest mortality was found in tilapia injected with Enterobacter sp., Listeria sp. and
Streptococcus sp. at a density of 109 CFU mL-1. However, the number of bacteria causing mortality in fish was approximately 10 4-108
CFU mL-1. All bacteria detected in the tilapia and channel catfish cultures were also known as putative pathogens in human.

Keywords: Bacteria colony, contaminant, channel catfish, pathogen bacteria, tilapia

INTRODUCTION in fish cultured in Asia. Meanwhile, pathogenic bacteria

were found in catfish including Escherichia coli (Zahran et
Tilapia (Oreochromis niloticus) and channel catfish al. 2016), Staphylococcus aureus, Salmonella typhi,
(Clarias batrachus) are two of the most widely distributed Pseudomonas aeruginosa, Vibrio cholera, and Shigella
aquaculture commodities worldwide. There has been a dysenteriae.
large increase in demand for tilapia and catfish, resulting in Gram-negative bacteria from the genera Aeromonas,
the increased production of both fishes through the method Flavobacterium, Pseudomonas, Francisella, and gram-
of high stocking density. However, the high stocking positive bacteria from the genera Streptococcus and
density method leads to rapid deterioration of water quality Lactococcus cause rapid high mortality in fish as soon as
and increased fish stress (Sebastião et al. 2015), resulting in 72 h after infection. Recently, there was an outbreak of fish
disease and pathogenic epizootics. disease in Thailand, causing a mortality rate of almost 90%
The existing pathogens in fish represent a serious (Dong et al. 2017a). Over 2015-2016, mass mortality in
problem and major concern for aquaculturists due to loss of tilapia and hybrid red tilapia (Oreochromis spp.) has been
income, reducing global aquaculture development (Lafferty investigated without determining the cause of death.
et al. 2015). Some of the pathogenic bacteria infecting However, histopathology analysis revealed that liver
tilapia include Aeromonas spp., Pseudomonas spp., damage had been found in the dead tilapia. Moreover,
Streptococcus spp., Vibrio spp., Enterococcus spp., Tilapia lake virus (TiLV) has also been detected in samples
Micrococcus spp., Staphylococcus spp., Plesiomonas spp., by using a scanning electron microscope (Dong et al. 2017b).
Moraxellaceae, and Enterobacteriaceae. Furthermore, Some of pathogenic bacteria in fish are facultative
Streptococcus spp. is a bacteria that causes high mortality (Nayak 2010) and able to survive in water for long periods
 HARDI et al. – Potential bacteria pathogen in fish 481

of time, thus making their presence difficult to prevent. Sheehan et al. (2009), and Hardi et al. (2011). All bacterial
Examination of pathogenic bacteria should be completed isolates were cultured in a phosphate-buffered broth base
regularly to anticipate the occurrence of a pathogen attack. with glucose, streaked on a blood agar plate, and examined
The success of disease control, including prevention and for their purity. Briefly, a suspension was made from the
treatment, is strongly influenced by the accuracy of disease cultures grown on blood agar plates by transferring a heavy
diagnosis in fish. inoculum of the culture to a tube containing 2 ml of
The present research aims to isolate, identify, and distilled water. Three drops of the suspension were placed
characterize pathogenic bacteria in tilapia and channel into a microcapsule using a Pasteur pipette. Strips were
catfish culture, providing updated information on the incubated at 37°C in a normal atmosphere. After 4 h of
development of disease-causing bacteria on both tilapia and incubation, reagents were added and strips were exposed to
channel catfish cultures. Our results also provide an early a strong light (100W for 10 s) for enzymatic activities
warning against the occurrence of disease outbreaks reading. Test results were then recorded, and the strips
informing an anticipated disease control and management were incubated again for 20 h. For the identification of
on the freshwater fish culture. Streptococcus, the interpretation of the test results obtained
at 4 and 24h after incubation species was based on the
profile index and table issued by the manufacturer.
MATERIALS AND METHODS
Antibiotic sensitivity
Sample preparation Bacterial sensitivity test against antibiotics was
Samples of water and fish feces were collected from completed using seven types of commercial antibiotic,
culture ponds containing tilapia (Oreochromis niloticus) namely: nitrofurantoin (Nitro), ciprofloxacin (Cipro),
and catfish (Clarias batrachus) in Samarinda City, East oxytetracycline (Oxy), chloramphenicol (Chlo), nalidixic
Kalimantan, Indonesia. Before being used, samples were acid (Na), gentamicin (Gent), and norfloxacin (Nor) using
cooled to 4oC. the inhibition zone method. The diameter of the zone of
inhibition appearing on the agar plate was measured to
Isolation and bacteria identification determine antibiotic resistance. Using this method, bacteria
All samples were cultured on nutrient agar media (NA, were inoculated in a liquid medium of trypticase soy broth
Oxoid), trypticase soy agar (TSA, Oxoid), and brain heart (TSB, Oxoid) followed by reading on solid medium TSA.
infusion agar (BHIA, Oxoid). A pure isolate was obtained After 5 min, antibiotic paper was placed on the medium
by reisolating a single bacterial colony onto each respected and incubated at 30oC for 24 h.
new media. Pure isolates were then used for colony
morphology and biochemistry identification and tested for Koch’s postulates test
their level of pathogenicity in tilapia. The Koch postulates test was performed according to
Jordan (1941), Kamiso et al. (2005), and Hardi et al.
Colony identification (2012), using tilapia. Tilapia is a common fish used for
In bacterial colony identification, the colony was bacterial pathogenicity testing, because tilapia has a
classified based on the colony color, form, and margin. constant health condition, a differential leukocyte consisting
According to Reynolds (2011), a bacterial colony has of neutrophils, monocytes, and lymphocytes of large size,
different morphological properties. Colony forms include making it easier for observation immune system of fish.
irregular, filamentous, rhizoid, and curled. Meanwhile, Bacteria were cultured in TSB medium at 30oC for 24h.
colony margin can be entire, filamentous, undulate, and After a total plate count (TPC) was performed to measure
lobate. Colony elevation can be categorized as raised, flat, the microbial density, bacteria were administered
convex, umbonate, and crateriform. The size of a bacterial intraperitoneally to tilapia with a dose of 10 3,5,7,9 bacterial
colony can be described as small, moderate, and large. cells per fish in triplicates. Control fish was injected with
Furthermore, the opacity of bacterial colony can be stated 0.1 mL sterile Phosphate Buffer Saline (PBS) per fish. All
as clear, opaque, translucent, and iridescent. fish were then reared in an aquarium with aeration and fed
with pellet at a rate of 3-5% of body weight, twice a day.
Biochemical identification After 7 days of rearing, cumulative mortality was
Biochemical identification of bacteria was performed calculated until day 7.
following the method of Indonesian National Standard, also
known as Standar Nasional Indonesia (SNI) No.
7303.1:2015, SNI No. 7545.3:2009 and SNI No. RESULTS AND DISCUSSION
8096.1:2015. The identification test includes gram staining,
a motility test using semi-solid medium, an oxidative- Bacteria colony
fermentative test, a catalase test, and a growth test on D- Bacterial isolates grown in TSA, NA, and BHIA
mannitol medium. medium in the first isolation and re-isolation are shown in
Figure 1. There were 37 bacteria colonies discovered in the
API test tilapia and catfish pond cultures (Table 1).
Bacteria identification using API 20 Strep and API 20 E BHIA is a rich medium, likely resulting in a higher
using the methods of Poutrel and Ryniewicz (1984), number of colonies successfully growing on it. From BHIA
482 B I O D I V E R S I T A S 19 (2): 480-488, March 2018

culture, we obtained 16 bacterial isolates. A total of 12 Pseudomonas sp., Streptococcus sp., Staphylococcus sp.
isolates grew in TSA medium and only nine isolates grew are obligate or facultative bacteria that can survive for long
in NA medium. periods in ponds if they found suitable hosts (Austin and
As seen from Table 1, the number of bacterial colonies Austin 2007).
isolated from tilapia and catfish ponds was 18 and 19
isolates, respectively. From the nine isolates obtained from
the water in tilapia’s pond, four isolates grew on BHIA,
three isolates on NA, and two on TSA. Meanwhile, from
fish feces, there were nine isolates: five grew on BHIA,
three on TSA, and only one on NA.

Bacteria identification
Using identification methods according to SNI 2009
and 2015, which includes gram, catalase, motility,
mannitol, fermentative test, O-F test, and growth in NaCl
medium, there were 37 bacteria isolates identified, of
which 22 isolates were gram-negative bacteria, and 15 A B
isolates were gram-positive. Similar to the previous
Figure 1. Bacterial isolates. A. First isolation in artificial NA
research by Marcel et al. (2013) in Malaysia, gram- medium, B. Re-isolated bacteria
negative is the most predominant bacteria found in tilapia
cultured. Generally, bacteria such as Aeromonas sp.,

Table 1. Characterization of bacterial colonies isolated from tilapia (Oreochromis niloticus) and catfish (Clarias batrachus) ponds based
on their form, color, and margin/edge of the colony

Source of sample Morphology of bacterial colony

Isolate code Medium
Tilapia pond Catfish pond Form Color/pigmentation Margin /edge
RA1 Water - TSA Irregular White Erose
RA2 Water - TSA Irregular White milk Erose
RA3 Water - NA Circular Translucent Entire
RA18 Water - NA Circular White milk Entire
RA14 Water - NA Circular White milk Entire
RA22 Water - BHIA Circular Creamy yellow Erose
RA23 Water - BHIA Circular Yellow Erose
RA24 Water - BHIA Irregular White Erose
RA30 Water - BHIA Circular White Erose
RA9 Feces - TSA Irregular Translucent Erose
RA10 Feces - TSA Circular White milk Erose
RA11 Feces - TSA Irregular White milk Erose
RA21 Feces - NA Circular Yellow Erose
RA28 Feces - BHIA Circular White milk Erose
RA29 Feces - BHIA Circular Translucent Erose
RA31 Feces - BHIA Irregular White milk Erose
RA32 Feces - BHIA Irregular White Erose
RA33 Feces - BHIA Irregular Translucent Erose
RA4 - Water TSA Circular Translucent Undulated
RA5 - Water TSA Circular Translucent Erose
RA6 - Water TSA Irregular Creamy yellow Erose
RA7 - Water TSA Circular White milk Undulated
RA8 - Water TSA Irregular Yellow Erose
RA15 - Water NA Circular White milk Erose
RA16 - Water NA Circular White milk Erose
RA17 - Water NA Circular White milk Erose
RA19 - Water NA Circular White milk Erose
RA25 - Water BHIA Circular White milk Erose
RA26 - Water BHIA Circular Creamy yellow Erose
RA27 - Water BHIA Circular Yellow Erose
RA12 - Feces TSA Irregular White milk Erose
RA13 - Feces TSA Irregular Translucent Erose
RA20 - Feces NA Circular Yellow Erose
RA34 - Feces BHIA Circular Yellow Entire
RA35 - Feces BHIA Circular White milk Erose
RA36 - Feces BHIA Circular White milk Erose
RA37 - Feces BHIA Circular Translucent Erose
 HARDI et al. – Potential bacteria pathogen in fish 483

Large colony,
circular form,
entire margin,
yellow

Small colony,
circular form,
erose margin,
white

Large colony,
circular form,
entire margin,
yellow

Figure 2. Bacterial colony forms in a variety of media

Based on the data presented in Table 2, there were All isolates from the Streptococcus group had the
seven genera of bacteria isolated from tilapia and catfish capability to degrade sodium pyruvate, hippuric acid,
ponds: Streptococcus sp. (13.5%), Staphylococcus sp. escullin, pyrrolidonyl arylamidase, α-galactosidase, β-
(24.32%), Pseudomonas sp. (10.81%), Enterobacter sp. glucuronidase, β-galactosidase but not L-arabinose and
(43.24%), Aeromonas sp. (10.81%), Neisseria sp. inulin (Table 3). Meanwhile, Enterobacter groups which
(10.81%), and Listeria sp. (10.81%). Marcel et al. (2013) were further tested using API 20E possessed two types of
also mentioned that Aeromonas hydrophila, glucose that cannot be degraded by all isolate, namely
Staphylococcus spp., P. aeruginosa, and Enterobacter trisodium citrate and urea (Table 4).
cloacae also existed in Kenyir Lake, Malaysia and found A. The present results of bacterial identification using
hydrophila and Staphylococcus sp. from Semantan River, biochemistry tests followed by API 20 Strep and API 20 E
where red tilapia is cultured in karamba floating nets. A tests is summarized in Table 5.
total of 19 isolate bacterial isolates were found in catfish Identification results of all the bacterial isolates using
ponds, dominated by Staphylococcus sp. and Enterobacter biochemistry tests, including API 20 E and API 20 Strep,
sp. However, Pseudomonas sp. (5%) and Listeria sp. (5%) revealed there were 14 bacterial species (Table 5)
were not found both in water or feces sample from tilapia including: Acinetobacter calcoaceticus (1 isolates),
pond. According to Gauthier (2014) and Boylan (2011), Aerococcus urinae (2 isolates), A. hydrophila (1 isolate),
bacterial groups Enterobacteriaceae, Streptococcus, Aerococcus viridans (1 isolate), Citrobacter freundii (5
Staphylococcus, Mycobacterium, Nocardia, Aeromonas, isolates), Enterobacter amnigenus (2 isolates), E. cloacae
Edwardsiella, Francisella, Pseudomonas, Vibrio, and (4 isolates), E. coli (3 isolates), Listeria sp. (1 isolate),
Yersinia are zoonotic bacteria in fish for human. Neisseria sp. (4 isolates), Pantoea spp. (1 isolate), P.
To confirm the type of bacteria that have been isolated aeruginosa (1 isolates), S. aureus (9 isolates), and
and identified, a follow-up test was conducted using API Streptococcus iniae (2 isolates). All 14 bacteria are not
20E, API 20 Strep. The results indicate that some bacteria only pathogens to fish but have also been reported causing
were able to hydrolyze some sugar (Table 3). Furthermore, zoonosis in human (Boylan 2011 and Gauthier 2015).
the bacterial group included in Enterobacteriaceae was According to Haenen et al. (2013), S. iniae is a zoonotic
identified by using API 20 E, while Streptococcus, pathogen in freshwater and marine fish aquaculture. In
Aeromonas, Pseudomonas groups were tested using API 20 addition, this bacterial invasive disease is carried by human
Strep (Tables 3 and 4). and can cause diseases in aquaculture, resulting in economic
losses in the tilapia aquaculture industry.
484 B I O D I V E R S I T A S 19 (2): 480-488, March 2018

Table 2. Characteristics of bacteria isolated from tilapia Table 3. Overview of Streptococcus bacteria characteristics using
(Oreochromis niloticus) and catfish (Clarias batrachus) ponds the API 20 strep test
using biochemical methods
Isolate code
Active ingredients
RA1 RA2 RA3 RA5 RA18

fermentative

Growth in
Sodium pyruvate + + + + +
Mannitol
Catalase
Gram

Motil

NaCl
Isolate Bacterial Hippuric acid + + + + +

O/F
code group Escullin + + + + +
Pyrrolidonyl arylamidase + + + + +
α-galactosidase + + + + +
RA1 + - - - F+ + Streptococcus β-glucuronidase + + + + +
RA2 + - - - F+ + Streptococcus β-galactosidase + + + + +
RA3 + - - - F+ + Streptococcus Alkaline phosphatase + + + - -
RA4 - + + + F+ + Enterobacter L-leucine aminopeptidase + + + - -
RA5 + - - - F+ + Streptococcus L-arginine + - + + +
RA6 - + + + F+ + Enterobacter D-ribose - + + + -
RA7 + + - + O+ + Staphylococcus L-arabinose - - - - -
RA8 - + + + O- - Enterobacter D-mannitol - + - - -
RA9 - + + + F+ + Enterobacter D-sorbitol - + - - -
RA10 - + + + F+ + Enterobacter D-lactose - + - - -
RA11 - + + + F+ + Enterobacter D-trehalose - + - + +
RA12 - + - - O- + Pseudomonas Inulin - - - - -
RA13 - + + + O- Enterobacter D-raffinose - - + - -
RA14 + + - + O+ + Staphylococcus Starch - + + + -
RA15 + + - + O+ + Staphylococcus Glycogen - + - - -
RA16 - + + + O- - Enterobacter
RA17 - + + + O- Enterobacter
RA18 + - - - F+ + Streptococcus
RA19 - + + + O- - Enterobacter Streptococcus sp. is a pathogenic bacterium in
RA20 + + - + O+ + Staphylococcus
freshwater fish. There are two main groups of
RA21 - + + + F+ + Enterobacter
RA22 - + + - O+ - Neisseria Streptococcus sp. which have pathogenic properties in fish,
RA23 - + + - O+ - Neisseria namely S. iniae and S. agalactiae (Hardi et al. 2011). The
RA24 - + + + F+ + Enterobacter infection of S. agalactiae has caused neonatal meningitis in
RA25 + + - + O+ + Staphylococcus human, and mastitis in cows (Elliott et al. 1990; Bohnsack
RA26 - + + - O+ - Neisseria et al. 2004; Lindah et al. 2005). In tilapia, these bacteria
RA27 + + - + O+ + Staphylococcus have caused whirling disease, exophthalmia, opacity, and
RA28 - + + + O- - Enterobacter purulence (Hardi et al. 2011). Besides fish, marine
RA29 + + - + O+ + Staphylococcus mammals, cows, horses, cats, and humans can host these
RA30 + + - + O+ + Staphylococcus
bacteria. S. agalactiae outbreaks are acute and have
RA31 - + + + O- - Enterobacter
RA32 - + + + F+ + Aeromonas previously caused 100% mortality in fish cultured by 14
RA33 - + + + F+ + Enterobacter days post-infection (Evans et al. 2006).
RA34 - + + - O+ - Neisseria Furthermore, Staphylococcus sp. is a gram-positive,
RA35 - + + + O- - Enterobacter non-spore, non-motile, facultative anaerobic
RA36 + + + - O- - Listeria chemoorganotrophic bacterium with a fermentative
RA37 + + + - O- - Staphylococcus metabolism (Holt et al. 1994). Staphylococcus sp. is a fish
pathogen that can be found in the intestine and feces of fish
(Caretto et al. 2005). In England, European sea brass
(Dicentrarchus labrax) has been infected by S. xylosus, S.
Further identification of commercial antibiotic chromogens, S. warneri, which was marked by a dark
sensitivity suggests that bacteria can be inhibited by body, ulceration, and necrosis either in fins or skin (Nizan
antibiotics such as Nitro = nitrofurantoin, Cipro: and Hammerschlag, 1993). Staphylococcus sp. is a
ciprofloxacin, Oxy: oxytetracycline, Chlo: chloramphenicol, dominant bacteria also found in tilapia from Kenyir Lake,
NA: nalidixic acid, Gent: gentamicin, and Nor: norfloxacin Malaysia (Zahra et al. 2004; Zahra et al. 2008; Marcel et al.
(Table 6). 2013).
The present study found that the antibiotic According to Pirie (1940), Listeria sp. belongs to the
nitrofurantoin effectively inhibited all the isolated bacteria order Bacillales, family of Listeriaseae and the Listeria
except S. aureus isolates RA 7 and RA 29. Meanwhile, A. genus, which can be found in both water and soil (Esteben
viridans was only sensitive to nitrofurantoin but not to et al. 2009). Listeria monocytogenes, found in freshwater,
other commercial antibiotics. According to Mohan et al. brackish, and marine fish (Novotny et al. 2004), can also be
(2017), the two of isolates A. viridans were resistant to found in harvested marine fish, presumably as a result of
nitrofurantoin, which is considered to be a first line drug in contamination during the handling process (Huss et al.
patients. 2000; Ariyanti 2010).
 HARDI et al. – Potential bacteria pathogen in fish 485

Table 4. Overview of Enterobacter bacteria characteristics using API 20E

Isolate code
Active ingredients
RA4 RA6 RA8 RA 9 RA 10 RA11 RA13 RA16 RA17 RA19 RA21 RA24 RA28 RA31 RA32 RA33 RA 35
2-nitrophenyl-ßD-galactopyranoside - + + + + - - + - + + + - + - + +
L-arginine + - - + + - + + - - + - + + + + -
L-lysine - + + - - - - - - - - + - - - - +
L-ornithine - + + + + - - + - - + + - + - + +
Trisodium citrate - - - + + - + + - - + - - + - + -
Sodium thiosulfate - - - - - - - - - - - - - - - - -
Urea - - - - - - - - - - - - - - - - -
L-tryptophane + - - - - - - - - - - - - - - - -
L-tryptophane - + + - - - - - - - - + - - - - +
Sodium pyruvate - - + + + - - - + - - - + - + + -
Gelatin - - - - - - + - - + - - + - + - -
D-glucose + + + + + + - + - - + + + - + + +
D-mannitol - + + + + - - + - - + + + + + + +
Inositol - - - + + - - - - - - - + - + + -
D-sorbitol - + + + + - - - - - - + + + + + +
L-rhamnose - + + - - - - + - - + + + + - - +
D-sucrose - + + + + - - + - - + + + + + + +
D-melibiose + + + + + + - + + - + + - + - + +
Amygdalin - - - + + - - + + - + - + + + + -
L-arabinose + + + + + + - + - - + + - + - + +
Oxidase - - - - - - + - - + - - - - - + -

Table 5. Bacteria identification from tilapia and catfish culture Table 6. Bacterial sensitivity to various antibiotics (mm)
ponds in Samarinda, East Kalimantan, Indonesia
Isolate code Nitro Cipro Oxy Chlor Na Gent Nor
Isolate code Bacteria RA 1 19 28 31 16 23 26 28
RA 1 Aerococcus urinae RA 2 16 0 0 0 0 7 0
RA 2 Aerococcus viridans RA 3 17 16 18 15 8 10 8
RA 3 Aerococcus urinae RA 4 10 16 17 15 8 11 0
RA 4 Citrobacter freundii RA 5 15 17 9 10 8 10 12
RA 5 Streptococcus iniae RA 6 12 25 17 16 18 13 26
RA 6 Escherichia coli RA 7 8 22 21 19 17 18 19
RA 7 Staphylococcus aureus RA 8 13 27 22 20 16 13 25
RA 8 Escherichia coli RA 9 11 22 12 11 10 13 10
RA 9 Citrobacter freundii RA 10 12 26 17 10 20 11 16
RA 10 Enterobacter cloacae RA 11 10 18 13 19 20 13 15
RA 11 Acinetobacter calcoaceticus RA 12 12 12 11 14 16 15 17
RA 12 Pseudomonas aeruginosa RA 13 11 10 17 17 14 12 21
RA 13 Citrobacter freundii RA 14 17 8 8 20 15 7 7
RA 14 Staphylococcus aureus RA 15 17 9 24 23 16 10 13
RA 15 Staphylococcus aureus RA 16 13 21 12 17 17 12 23
RA 16 Enterobacter amnigenus RA 17 14 12 11 13 12 9 8
RA 17 Enterobacter amnigenus RA 18 15 15 13 0 0 7 20
RA 18 Streptococcus iniae RA 19 17 17 20 19 15 10 22
RA 19 Escherichia coli RA 20 19 22 9 21 13 14 13
RA 20 Staphylococcus aureus RA 21 18 24 7 19 25 12 25
RA 21 Citrobacter freundii RA 22 12 14 17 15 15 13 12
RA 22 Niseria sp. RA 23 12 16 21 15 15 13 14
RA 23 Niseria sp. RA 24 10 17 20 16 16 12 13
RA 24 Citrobacter freundii RA 25 18 17 27 16 24 16 20
RA 25 Staphylococcus aureus RA 26 16 16 14 14 7 11 11
RA 26 Neisseria sp. RA 27 16 16 21 16 14 11 18
RA 27 Staphylococcus aureus RA 28 12 15 16 10 16 13 15
RA 28 Pantoea spp. RA 29 9 14 19 20 15 15 16
RA 29 Staphylococcus aureus RA 30 13 15 15 19 17 12 15
RA 30 Staphylococcus aureus RA 31 12 25 22 18 19 14 23
RA 31 Enterobacter cloacae RA 32 14 15 17 17 17 15 17
RA 32 Aeromonas hydrophila RA 33 10 16 13 16 17 12 20
RA 33 Enterobacter cloacae RA 34 11 22 21 16 10 10 14
RA 34 Neisseria sp. RA 35 10 24 19 16 16 12 19
RA 35 Enterobacter cloacae RA 36 14 17 7 19 20 15 18
RA 36 Listeria sp. RA 37 14 15 12 16 24 16 17
RA 37 Staphylococcus aureus Note: Nitro = nitrofurantoin, Cipro: ciprofloxacin, Oxy:
oxytetracycline, Chlo: chloramphenicol, NA: nalidixic acid, Gent:
gentamicin, Nor: norfloxacin
486 B I O D I V E R S I T A S 19 (2): 480-488, March 2018

Enterobacter sp. belongs to order Enterobacteriales, 1990, Baya et al. found C. freundii from Atlantic salmon
the family Enterobacteriaceae, and genus Enterobacter. (Salmo salar) in Spain and the USA, while Sans (1991)
Besides living as opportunistic pathogens in fish identified the bacteria in Cyprinus carpio from India
(Rajasekaran 2008), Enterobacter sp. can be used as (Karunasagar and Pari 1992).
indicator of waste pollution because of their presence in Moreover, the percentage of fish mortality caused by
humans, animals, water, soils, plants, insects, and Enterobacter sp. was 90% in the present study (Table 6).
processed products (Davidson et al. 2000; Neto et al. This finding is in line with Sekar et al. (2008), who
2003). Previous research performed in Egypt by Hasan et reported that Enterobacter sp. caused 100% mortality in
al. (2012) reported that 9 species of bacteria of Mugil cephalus six days after bacterial injection. The
Enterobacteria, E. coli, Salmonella arizonae, Citrobacter previous research also suggests that tilapia injected
braakii, Enterobacter sakazakii, C. freundii, Rautella intraperitoneally with Streptococcus agalactiae (density
ornithinolytica, Klebsiella ozaenae, E. cloacae, and 104 CFU mL-1) experienced mortality of up to 48% at day 7
Proteus vulgaris have been found in tilapia, with an (Hardi et al. 2011). Similar to previous research, the current
infection prevalence of 2.7-27 %. Noor El-Deen et al. findings revealed that a bacterial density of 10 3 CFU mL-1
(2010) and Olurin et al. (2006) revealed that a fish infected resulted in 40% mortality in tilapia sampled. Additionally,
with Enterobacter exhibited pale internal organs such as Staphylococcus sp. caused mortality of up to 80% at a
liver and kidney, and caused anemia, intestinal congestion, density of 109 CFU mL-1, with an average LD 50 at a
ulceration of the anus accompanied by mucus and bleeding density of 104.40 CFU mL-1 of bacteria. According to
in its infected external organs. Kamiso et al. (2005), bacteria with an LD50 below 10 5
Dharma (1982) stated that C. freundii disrupt the CFU mL-1 has been categorized as being pathogenic.
internal organs of fish, including intestines, liver, and
kidneys. The intestines of an infected fish blacken and Table 6. The percentage of cumulative mortality in tilapia
become mushy and juicy in consistency. Disruption of liver (Oreochromis niloticus) injected at varying bacterial densities
and bile functions is caused by increased liver activity from
Density Day
processing and neutralizing metabolites and toxic Bacteria
(CFUmL-1) 1 2 3 4 5 6 7
substances.
Citrobacter freundii 103 0 0 10 10 20 20 20
According to Novotny et al. (2004), bacteria that was 105 0 10 10 20 30 30 30
found both in feces and water in tilapia and catfish ponds 107 20 30 30 30 30 40 50
are categorized as pathogenic to humans. The occurrence 109 30 30 40 40 50 60 60
of pathogenic bacteria such as Mycobacterium spp., S.
iniae, Photobacterium damselae, Vibrio alginolyticus, Enterobacter sp. 103 0 0 0 10 20 20 20
Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholera, 105 10 10 20 30 30 30 30
E. coli, Aeromonas spp., S. aureus, Listeria monocytogenes, 107 20 40 50 50 50 80 80
Clostridium botulinum, Clostridium perfringens, 109 20 40 50 60 80 80 90
Campylobacter jejuni must be anticipated in order to
Staphylococcus sp. 103 0 0 0 0 20 30 30
reduced economic loss in cultured freshwater fish and to 105 20 20 20 40 40 50 60
prevent their transmission to the humans consuming these 107 0 20 30 40 60 60 60
fish. 109 10 20 30 60 70 80 80

Koch’s postulate test Listeria sp. 103 0 0 10 10 10 10 10

To examine each bacterium’s pathogenicity in fish, we 105 20 30 30 30 30 30 50
performed Koch’s postulate test following the protocol of 107 60 60 60 60 60 60 80
Jordan (1941), Kamiso et al. (2005), and Hardi et al. 109 50 50 60 70 70 70 90
(2012). The test revealed that Streptococcus sp., C. Streptococcus iniae 103 10 10 20 20 20 40 40
freundii, Staphylococcus sp., Enterobacter sp., 105 20 20 30 50 50 60 60
Pseudomonas sp., Neisseria sp. and Listeria sp. caused 107 30 30 40 50 60 70 70
varying degrees of mortality in tilapia. The highest 109 30 40 50 60 80 80 90
mortality was found in tilapia injected with Enterobacter
sp., Listeria sp., and Streptococcus sp. at a density 109 CFU Pseudomonas sp. 103 0 10 10 10 10 10 10
mL-1 (Table 3). However, the number of bacteria causing 105 0 10 20 20 20 30 30
mortality in fish was approximately 104-108 CFU mL-1 107 0 30 40 50 50 50 50
109 20 30 30 40 50 60 60
bacterial density. According to Angka et al. (2002),
bacteria that caused mortality at a density above 105 CFU Aeromonas 103 0 10 10 20 20 30 30
mL-1 was categorized as being less pathogenic. hydrophila 105 0 10 20 20 20 30 40
Citrobacter freundii is a bacterial pathogen that caused 107 0 30 30 30 40 40 45
109 20 30 30 40 50 60 60
60% mortality in tilapia fish sampled in the present study
(Table 6). This finding is in line with a previous report by Neisseria sp. 103 0 0 0 0 0 0 0
Jeremić, et al. (2003) who isolated C. freundii from all sick 105 0 0 0 10 10 10 10
and dead Cyprinus carpio. Previously, Sato et al. (1982) 107 0 0 0 10 20 30 30
also indicated that C. freundii caused mortality in fish. In 109 20 20 30 30 30 30 30
 HARDI et al. – Potential bacteria pathogen in fish 487

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