Resistance Test of Vibrio sp. From Milkfish (Chanos chanos) in

Fishpond Jabon Sidoarjo to Heavy Metals and Antibiotics

Pratikah Verdianti

, Fitriyati Mukhlishoh

, Tuty Putri

,and Ayu Puspitasari

Forensics Science, Postgraduate School, Universitas Airlangga, Jl. Airlangga No. 4 -6, Surabaya, East Java, Indonesia

Study Program of Medical Laboratory Technology, Health Polytechnic Surabaya, Surabaya, Indonesia

Keywords: Vibrio sp, Resistance test, Lead (Pb), Cadmium (Cd), Tetracycline antibiotic, Chloramphenicol antibiotic.

Abstract: The milkfish (Chanos chanos) obtained from Fishponds Jabon Sidoarjo contained heavy metals such as Pb

and Cd. Heavy metals pollution can be controlled by bioremediation. The objective of this research is to

determine the resistance of Vibrio sp isolated from milkfish to heavy metals Pb, Cd, at various

concentrations and to know the resistance of these bacteria to antibiotics such as tetracycline and

chloramphenicol. Resistance tests of Vibrio sp were performed using dilution and pour plate method. The

Nutrient agar media used in this study contained heavy metals PbCl

and CdCl

with concentrations of 5

ppm, 10 ppm, 25 ppm. Heavy metals-resistant Vibrio sp was tested its antibiotic resistance with diffusion

method using antibiotic disks such as Tetracycline 30 μg and Chloramphenicol 30 μg. The results showed

that Vibrio sp was resistant to heavy metals Pb and Cd at all concentrations. Antibiotic sensitivity test using

chloramphenicol 30 μg showed that Vibrio sp was resistant to chloramphenicol at concentration of 5ppm.

Pb-resistant Vibrio sp was shown resistant to chloramphenicol at concentration of 10 ppm and 25 ppm. Pb-

and Cd-resistant Vibrio sp were shown resistant to Tetracycline 30 μg at all concentrations.

1 INTRODUCTION

In 2014, there were approximately 79% rivers with

status of slight, moderate, and severe pollution,

while in 2015 there was increasing number of

polluted rivers. One of polluted rivers in East Java

was Porong River in Kabupaten Sidoarjo due to

disposal of lapindo mud by Lapindo Brantas Inc.

This led to reduction of ecosystem caused by heavy

metals contamination such as Lead (Pb) and

Cadmium (Cd).

Kholidiyah (2010) stated that concentrations of

heavy metals in the water of Porong river and

lapindo mud exeeded the standard level, showing

Lead (Pb) at concentration of 7,2876 mg/L in

lapindo mud and 0,6949 mg/L in the water of

Porong river, whereas Cadmium (Cd) contained in

lapindo mud were at concentration of 0,3063 mg/L

and of 0,0271 mg/L in the water of Porong river.

This caused pollution of nearby fishponds.

Both Lead (Pb) and Cadmium (Cd) are heavy

metals that can accumulate inside the body of living

creatures especially fishes in water contaminated

with heavy metals, potentially become toxic. If those

fishes are consumed by human, toxic will

accumulate inside tissues of human body and

endanger their health (Nuhman, 2003) One of the

known contaminated fishes is Bandeng or Milkfish

(Chanos chanos), a brackish water fish commonly

bred in fishponds.

Larasati (2015) in her research stated that

milkfish bred in fishpond Jabon Sidoarjo located at 5

km, 10 km, and 15 km away from lapindo mud

showed average concentration level of Pb of 1,0985

mg/kg, 1,3408 mg/kg, and 1,0839 mg/kg. Based on

those results it could be concluded that milkfish was

contaminated with Pb at concentration exceeding

standard of heavy metal concentration according to

SNI 7387:2009 which is 0,3 mg/kg. Screening test to

detect heavy metals contained in milkfish living in

fishpond Jabon Sidoarjo (2017) showed the result as

much as 4,82 ppm and 6,11 ppm of Pb, while Cd

was as much as 2,63 ppm and 3,24 ppm.

One of methods to overcome contamination

caused by heavy metals is by bioremediation, a

technique using certain microorganisms to eliminate

effect of contaminants (toxic substances), in order to

reduce contaminating substances (Sitanala, dkk.,

2008). Microorganisms which can be used in

bioremediation are those resistant to heavy metals.

Verdianti, P., Mukhlishoh, F., Putri, T. and Puspitasari, A.

Resistance Test of Vibrio Sp from Milkish (Chanos chanos) in Fishpond Jabon Sidoarjo to Heavy Metals and Antibiotics.

DOI: 10.5220/0007543203710375

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 371-375

ISBN: 978-989-758-348-3

371

After screening test of bacterial isolation from

milkfish (2017) was performed, the result showed

that Vibrio sp was contained inside the milkfish.

Vibrio sp, according to Utomo (2016) is indigenous

bacteria naturally found in fish.

Ayu (2013) stated that if certain microorganisms

contain any substances resistant to heavy metals and

antibiotics, they will negatively affect to human’s

health as treatment failure. Therefore, it is necessary

to perform resistance test using antibotics, a

chemical substance produced by certain

microorganisms that inhibit growth or kill other

microorganisms. (Simangunsong, et al., 2015).

Antibiotics often used in medical treatment are

tetracycline with wide spectrum for certain

indication from bacteria (BPOM, 2015) and

chloramphenicol which is a preferred antibiotic for

treating infection of gram-positive and gram-

negative bacteria (Uddin, et al., 2016).

Based on facts above, It was necessary to

perform a study about resistance of Vibrio sp

isolated from Milkfish (Chanos chanos) in fishpond

Jabon Sidoarjo to heavy metals such as Lead (Pb)

and Cadmium (Cd) and to antibiotics such as

Tetracycline and Chloramphenicol.

The aim of this study was to identify the

resistance of Vibrio sp to Pb and Cd at

concentrations of 5 ppm,10 ppm and 25 ppm, and to

identify the resistance of heavy metals-resistant

Vibrio sp to Tetracycline and Chloramphenicol.

2 RESEARCH METHOD

2.1 Type of Research

This study used explorative descriptive method.

2.2 Place and Time

This study took place in Microbiology Laboratory of

study program of Medical Laboratory Technology,

Health Polytechnic Surabaya starting from January

until June 2017.

2.3 Tools and Materials

Tools used in this study were autoclave, incubator,

inoculating loop, spirit burner, laboratory tripod,

erlenmeyer flask, petri dish, laboratory cottons,

analytical balance, glass stirring rod, sterile alcohol

swab, pH-indicator papers, graduated cylinder,

laboratory funnel, tweezers, rubbers, alumunium

foil, volumetric flask, beaker glass, and watch glass.

Materials used in this study were isolates of

Vibrio sp from milkfish, NA (Nutrient Agar) media,

PbCl

, CdCl

, NB (Nutrient Broth) media, MH

(Mueller Hinton Agar) media, Mc Farland

standards, antibiotic sensitivity disks Tetracycline 30

µg and Chloramphenicol 30 µg.

2.4 Procedures

2.4.1 Isolatioan of Bacteria from Milkfish

Milkfish firstly was cleaned from bones and mashed.

Serial dilution of fish samples then was performed

using saline solution to create dilution of 10

-3

. The

diluted samples were inoculated on Mac Conkey

Agar and incubated on 37

C for 24 24 hours.

Colonies grown on Mac Conkey Agar were picked

off and inoculated on Eosin Methylene Blue Agar

media, incubated on 37

C for 24 hours. Colonies

grown on EMB media were inoculated on Triple

Sugar Iron Agar media, incubated on 37

C for 24

hours. Colonies on TSIA media were further

identified using IMViC test (Indol, Methyl Red,

Voges Proskauer and Citrate) and Semi solid.

2.4.2 Resistance Test to Heavy Metals

Isolates of Vibrio sp were diluted until 10

-5

then

poured into NA-PbCl

and NA-CdCl

media with

concentrations used were 5 ppm, 10 ppm and 25

ppm. In addition, NA-PbCl

and NA-CdCl

media

were used as negative controls in the absence of

vibrio sp isolates. After 24 hours of incubation, the

grown isolates were shown to be resistant to Pb and

Cd, and negative controls media. Then, CFU

(Colony Forming Unit) was determined by

comparing the number of colony in media test amd

that of negative control media.

2.4.3 Resistance test to Antibiotics

Resistance tests of Vibrio sp to antibiotics were

performed by inoculating heavy metals-resistant

bacteria on MH (Mueller Hinton) media, putting

tetracycline disk and chloramphenicol using sterile

tweezers on the surface of media inoculated with

bacteria, and incubating those media for 24 hours.

Transparent zones were measured and results were

interpreted.

ICPS 2018 - 2nd International Conference Postgraduate School

372

3 RESULT

3.1 Resistance Test to Heavy Metals

Resistance tests of Vibrio sp to heavy metals were

performed by determining the viability of Pb- and

Cd-resistant Vibrio sp. The CFU/ml of Vibrio sp

isolates treated with Pb (Table 1) showed constant

number at all concentrations, while the CFU/ml of

Vibrio sp isolates treated with Cd showed certain

decline at concentration of 5 ppm, 10 ppm and 25

ppm (Table 1).

Isolates of Vibrio sp from milkfish were able to

grow on Pb- and Cd- containing media with

concentration of 5 ppm, 10 ppm, and 25 ppm (Figure

1). According to Zulaika et.al (2012), bacteria

isolated from heavy metals-contaminated

environment were resistant to surrounding heavy

metals.

Figure 1: Result of inoculation of Vibrio sp to Pb and Cd

with concentration of 5 ppm (a), 10 ppm (b), 25 ppm (c).

Table 1: Result of resistance test of Vibrio sp to Pb and

Cd.

3.2 Antibiotic Sensitivity Test

Result of antibiotic sensitivity test shown in Figure 2

and Table 2 showed that Pb-resistant Vibrio sp was

resistant to Tetracycline 30 µg at all concentrations

of heavy metals and was resistant to

Chloramphenicol at Pb concentration of 10 ppm and

25 ppm. Meanwhile, Pb-resistant Vibrio sp was

susceptible to Chloramphenicol 30 µg at Pb

concentration of 5 ppm.

(c)

Figure 2: Result of antibiotic sensitivity test of Pb-

resistant Vibrio sp.

Concentrations

of heavy metals

Pb Cd

CFU/ml

5 ppm 13,55 x 10

30,0 x 10

10 ppm 13,3 x 10

16,55 x 10

25 ppm 13,7 x 10

12,2 x 10

(

)

(

)

(c)

(a) (b)

Resistance Test of Vibrio Sp from Milkish (Chanos chanos) in Fishpond Jabon Sidoarjo to Heavy Metals and Antibiotics

373

Table 2: Result of antibimicrobial resistance test of Pb-

resistant Vibrio sp.

Inhibition zone

diameter of

Tetracycline

30 µg

Inhibition zone

diameter of

Chloramphenico

l 30 µg

5 ppm

17 mm,

resistant

29 mm,

susceptible

10 ppm

18 mm,

resistant

16 mm,

resistant

25 ppm

16 mm,

resistant

15 mm,

resistant

Antibiotic sensitivity test of Cd-resistant Vibrio

sp to Tetracycline 30 µg and Chloramphenicol 30 µg

using Cd at concentration of 5 ppm, 10 ppm, and 25

ppm showed that Cd-resistant Vibrio sp was resistant

to Tetracycline 30 µg and Chloramphenicol 30 µg at

all concentrations of Cd (Table 3).

Table 3: Result of antibiotic sensitivity of Cd-resistant

Vibrio sp.

Inhibition zone

diameter of

Tetracycline

30 µg

Inhibition zone

diameter of

Chloramphenico

l 30 µg

5 ppm

6 mm,

resistant

17 mm,

susceptible

10 ppm

6 mm,

resistant

15 mm,

resistant

25 ppm

6 mm,

resistant

15 mm,

resistant

4 DISCUSSIONS

4.1 Resistance Test to Heavy Metals

Vibrio sp was able to grow in such condition by a

mechanism called biotransformation.

Biotransformation is a process of enzyme production

possessed by microorganisms through a chemical

alteration of pollutants in order to modify toxic

pollutants. This process will lead to biodegradation

process which is the ability of microorganisms to

cleave chemical structure of toxic pollutants into

noncomplex substances with low toxicity (Perdana,

2012). Biodegradation is classified into two types,

namely extracellular and intracellular. Extracellular

mechanism is the ability of bacteria to detoxify the

effect of heavy metals with the availability of

polyphosphate precipitates or by forming

nonspecific bond with extracellular polysaccharides

or natural polymers in the cell wall whereas

intracellular mechanism of heavy metals is

unactivated through precipitation by polyphosphate,

binding to Metallothionein (MT), and efflux system

(Jaroslawiecka and Seget, 2014).

4.2 Antibiotic Sensitivity Test

Based on the results above, Pb and Cd-resistant

Vibrio sp were all resistant to Tetracycline 30 µg,

shown by inhibition zone diameters less than 18

mm. According to Byarugaba (2009) resistance can

naturally occur because microorganisms are not

easily affected by antibiotics. This statement was

supported by Johnson et.al (2011) stating that

mechanisms of antimicrobial are classified into two,

namely nongenetic mechanism caused by loss of

specific targeted structures and genetic mechanism

caused by chromosomal/ extrachromosomal

resistance in bacteria.

In addition to Tetracycline, chloramphenicol 30

µg was also used for antibiotic sensitivity test. Based

on the results above, it was found that Pb-resistant

Vibrio sp was resistant to Chloramphenicol at

concentration of 10 and 25 ppm and was resistant to

Cd at all concentrations, shown by inhibiton zone

diameters less than 19 mm. In contrast, Pb-resistant

Vibrio sp was susceptible to Chloramphenicol at

concentration of 5 ppm, shown by inhibition zone

diameters more than 19 mm. This was caused by the

wide-spectrum ability of antibiotics to effectively

inhibit growth of gram-negative bacteria by blocking

the activity of enzyme peptidyl transferase. This

ICPS 2018 - 2nd International Conference Postgraduate School

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enzyme plays a role in the process of forming

peptide bonds between new amino acids attached to

tRNA and the last amino acids which are still

developing in the bacteria. As the result, protein

synthesis in bacteria will be completely interrupted

(Pratiwi, 2008).

Gram-negative bacteria are more resistant to

drugs compared to gram-positive bacteria due to

efflux system possessed by gram-negative bacteria.

This system allows accumulated drugs inside cells to

be carried out, enabling drug concentration to be

reduced. The antibiotic sensitivity test showed that

bacteria can modify themselves to reduce effectivity

of certain drugs. As the result, the bacteria can

survive living and reproduce themselves to be more

endangering (Dwyana and Fahruddin, 2012).

5 CONCLUSIONS AND

SUGGESTIONS

5.1 Conclusions

According to study above, it could be concluded

that:

1. Vibrio sp was resistant to heavy metals such as

Pb and Cd at concentration of 5 ppm, 10 ppm,

and 25 ppm, suggesting that the bacteria could

survive living in the water regardless the

availability of Pb dan Cd.

2. Antibiotic sensitivity test showed that Pb-

resistant Vibrio sp was resistant to Tetracycline

30 µg at all concentrations but susceptible only

to Chloramphenicol 30 µg at concentration of 5

ppm. Antibiotic sensitivity test showed that Cd-

resistant Vibrio sp was resistant to Tetracycline

30 µg and Chloramphenicol 30 µg at all

concentrations.

5.2 Suggestions

1. Society should not excessively consume milkfish

due to side effect of heavy metals contained in

water where it lives and due to bacteria in it

which are resistant to heavy metals.

2. We suggest that the next researchers perform a

study using Pb and Cd with higher concentration

and do resistance test using different strains of

bacteria, heavy metals, and antibiotics.

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