Occurrence of Pathogenic Bacteria in Blood Cockles,

Anadara granosa

Nurin Syakirin Jantan, Zunita Zakaria, Saleha Abdul Aziz and Fuad Matori

Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Keywords: Cockles, Pathogenic Bacteria, Antibiotic Susceptibility Test, Multidrug Resistance.

Abstract: Anadara granosa (blood cockles), also known as kerang is a very popular seafood in Malaysia and South

East Asia. In 2015, there has been a drastic reduction in the harvest and one of the main reasons is due to

the deteriorating water quality in the cockles' breeding environment. Thus, these cockles are exposed and at

high risk of being contaminated by pathogenic microorganism because they are filter-feeder organism. Most

of the researches on Anadara granosa have focused on only few selected organisms. The overall

microbiological assessment of the cockles is lacking therefore this study aimed to determine the types of

pathogenic bacteria in blood cockles, Anadara granosa and their antibiotic susceptibility pattern. Thirty

pooled sample of Anadara granosa were purchased from 15 wet markets and supermarkets within Klang

Valley. All samples were subjected to isolation and identification using standard conventional method. A

total of 85 isolates were successfully isolated and all were gram negative bacteria. Antibiotic susceptibility

test was performed for the different types of bacteria obtained. All isolates were found to be resistant to

Ampicilin (10 μg) and were sensitive to Trimethoprim/sulfamethoxazole (25μg). In conclusion, this study

showed that cockles are exposed to highly pathogenic bacteria and there is presence of antibiotic resistance.

1 INTRODUCTION

Anadara granosa is shellfish members of the Class

Bivalvia, mollusks that enclosed between two shells

which are closed together and joined together with

elastic hinge ligament (Sauders Veterinary

Dictionary, 2nd Edition. Malaysia produced

100,000 tons of cockles for both local consumption

and export. The cockles are harvested from the

coastlines especially in Selangor, Perak and Johor.

They usually distributed at tidal mudflat area with

low oxygen content due to presence of hemoglobin

that have ability to retain high oxygen content.

Cockles are exposed and at high risk of

contaminated by multiple organism, for instance

bacterial, viral and toxin-producing dinoflagellates

because they are filter-feeder organism such as

phytoplankton, zooplankton, bacteria, viruses and

inorganic materials (Burkhardt & Calci 2000;

Rippey, 1994). Consumption of cockles that is

harvested from contaminated area can cause illness

to human.

According to previous studies, it reveals that

aquatic environment is a reservoir for antibiotic

resistance due to frequent usage of antimicrobial and

antibiotic contamination (Samuel et al. 2016; Huang

et al. 2001). Thus, presence of pathogenic bacteria

together with multiple antibiotic resistances found in

aquaculture product will become a threat to public

health. This study was conducted to determine the

types of pathogenic bacteria in blood cockles and

determine the antibiotic resistance of selected

isolated bacteria.

2 MATERIALS AND METHODS

2.1 Sample and Data Collection

Thirty samples of Anadara granosa were purchased

from 15 wet markets and supermarkets. The shells

were rinsed by 70% alcohol and the meat was

removed aseptically. Three grams of the sample was

homogenized with 30ml of peptone water by

stomacher for 2minutes.

268

Jantan, N., Zakaria, Z., Aziz, S. and Matori, F.

Occurrence of Pathogenic Bacteria in Blood Cockles, Anadara granosa.

DOI: 10.5220/0008888602680272

In Proceedings of the 7th International Conference on Multidisciplinary Research (ICMR 2018) - , pages 268-272

ISBN: 978-989-758-437-4

2.2 Methods

2.2.1 Isolation of Vibrio sp

The shell of Anadara granosa was cleaned using

70% alcohol. The meat was removed from the shells

and into 3g portions. The samples were then

homogenized with 30ml of alkaline peptone water

(Oxoid CM1028B) using stomacher for 2 minutes.

The homogenized sample was then incubated at 30

○

C for 24 hours under aerobic condition. After the

enrichment process, a lapful of the enriched sample

was streaked onto TCBS (CONDA). The plates

were incubated at 30

○

C for 24 hours under aerobic

condition.

2.2.2 Isolation of Bacteria Other than Vibrio

The shell of Anadara granosa was cleaned using

70% alcohol. The meat was removed from the shells

and into 3g portions. The samples were then

homogenized with 30ml of peptone water (Oxoid

CM009) using stomacher for 2 minutes. After

homogenization, a lapful of each homogenized

sample was streaked onto Blood agar (Oxoid

CM0055) and MacConkey Agar (Oxoid CM1169).

The plates were incubated at 30

○

C for 24 hours

under aerobic condition.

2.2.3 Identification of Vibrio sp

Presumptive Vibrio sp exhibiting green and yellow

colonies on TCBS agar were selected and Gram

stained. These colonies were sub-cultured into

Triptych Soy (TSA) agar (BD #221283) in order to

obtain pure culture. The cultures were subjected to

series of biochemical test including oxidase test,

ability to growth at different NaCl concentration,

Voges–Proskauer test (VP), Lysine Decarboxylase

(LDC) and Ortho-Nitrophenyl-β-Galactoside

(ONPG) or species identification.

2.2.4 Identification of Bacteria Other than

Vibrio sp

Each different type of colony was picked and sub

cultured into Blood agar in order to obtain pure

colonies for identification. Gram staining

(Appendix A) was performed and series of

biochemical tests for gram negative and gram

positive were carried out. Biochemical tests include

blood broth, 6.5%NaCl, bile, lactose, sorbitol and

trehalose for Gram positive bacteria. Gram negative

bacteria were subjected to triple sugar iron (TSI),

Sulphide-Indole-Motility (SIM), urea and citrate 2.1

Isolation and identification.

2.2.5 Antibiotic Sensitivity Test

Susceptibility of the obtained bacteria to selected

antibiotics was tested on Mueller Hinton agar (MHA)

plates by the disc diffusion method according to

Bauer et al. (1966). One colony from pure culture was

emulsified in sterile saline solution until the turbidity

was match with standard 0.5 MacFarland solutions. A

sterile swab was dipped into the bacterial suspension

and then streaked over the entire surface of Mueller-

Hinton agar and Blood agar. Six antibiotic discs

Oxoid, were aseptically placed on the swabbed plates.

The antibiotics discs used include ampicillin (10μg),

erythromycin (15μg), tetracycline (30μg),

enrofloxacin (5μg), gentamicin (10μg), trimethoprim

and sulfamethoxazole (25μg), Anti biotic disc used in

this study were antibiotics that commonly used in

aquaculture as well as in human medicine. The plates

were incubated at 30°C for 24 h and the clear zone

formed around the discs was measured by using

caliper. The growth inhibition zone was compared

with zone-size interpretative table as in (CLSI, 2010).

3 RESULTS

The overall isolated bacteria in 30 different wet

markets and supermarkets around Klang Valley

revealed that a total of 85 isolates were successfully

isolated and representing 13 different types of

bacteria species (Figure 1). The study showed that

Aeromonas spp (23%) was the most frequently

isolated bacteria from cockles followed by Proteus

mirabilis (20%), Vibrio alginolyticus (15%), Vibrio

parahaemolyticus (6%), Photobacterium damsel

(6%), Vibrio cholera (5%), Chromobacterium sp.

(5%), Proteus vulgaris (2%), Salmonella spp (2%),

Klebsiella pneumoniae (2%), Plesiomonas

shigelloides (2%) and E.coli (1%).

Antibiotic susceptibility of the isolates was

performed using 6 antibiotics ranging from broad-

spectrum antibiotics and narrow spectrum. All of

the isolates showed resistance to at least one

antibiotic. Three isolates were multidrug resistance

as they are resistant to more than three types of

antibiotics from different classes. The bacteria

isolates showed the highest percentages of resistance

towards ampicillin (68%), followed by erythromycin

(37%), tetracycline (21%), enrofloxacin (16%),

gentamicin (11%) and trimethoprim/

sulfamethoxazole (11%). Isolates showed most

Occurrence of Pathogenic Bacteria in Blood Cockles, Anadara granosa

269

resistant towards ampicillin and most were sensitive

to trimethoprim/ sulfamethoxazole (Figure 2).

Figure 1: Types of bacteria isolated from blood cockles.

Figure 2: Percentage of resistant bacteria against different

antibiotics.

4 DISCUSSION

All isolated bacteria consisted of gram negative

bacteria. This concurs with the results obtained by

Santos et al. (2010), which stated that majority of

bacteria within marine environment are gram

negative bacteria. To date, very limited studies have

been carried out on the microbiology assessment and

antibiotic resistance in bivalves in Malaysia. One

of those studies revealed that 93% of the isolated

bacteria were gram negative bacteria (Ahmad,

2014).

Bacteria obtained from this study can be

categorized into 2 family groups which are

Vibrionacea and Enterobacteriae. These two groups

can be easily differentiated by oxidase test for which

the Vibrionaceae will give positive result. These two

groups of family are mostly pathogens that usually

cause gastroenteritis in human. Aeromonas is the

most abundant isolated bacteria in this study.

Gastrointestinal infections caused by Aeromonads

are mostly self-limiting, and antibiotic therapy is

required only in chronic cases of immunosuppressed

hosts (Igbinosa, 2012). The least isolated bacteria

were from family of Enterobacteriaceae which are

E.coli (1%), Salmonella spp (2%), Klebsiella

pneumonia (2%), Plesiomonas shigelloides (2%),

Proteus vulgaris (2%) and Proteus mirabilis (20%).

Increase in bacterial contamination at beaches along

many coastlines usually occurred during heavy

rainfall or rainy season (Gregory, 2009). In this

study, sampling was done on the dry season in the

West Coast of Peninsular Malaysia, therefore it is

predicted more contaminants might be seen if it is

the rainy season.

According to Letchumanan (2014), Malaysia is

one of the Asian countries that often suffer food

borne outbreaks mainly caused by Vibrio sp. Other

countries include Japan, India, China, Taiwan and

Korea. Three members of the Vibrio genus were

isolated in this study were Vibrio alginolyticus,

Vibrio cholera and Vibrio parahaemolyticus. This

finding is in agreement with the studies of Ahmad

(2014) and Thompson (2014) which stated that

Vibrio sp is the most dominant genus present in

cockles. Vibrio sp have unique ability whereby they

are hardy organism as they able to withstand harsh

environment. They can be found in a wide range of

environment; from estuaries, coastal, marine water

and even sediment. In a previous study conducted

by Wan and Nor (2004) on bacterial quality of some

shellfish revealed that Vibrio spp. are commonly

isolated from cockles compared to other shellfish.

Some limitation on isolation and identification of

bacterial pathogens by using conventional methods

is lack of sensitivity as stated in Law et.al, 2014 and

this may lead to false negative e results. In addition,

marine organisms might occur in a state which is

viable but non-cultural. In this study approximately

ICMR 2018 - International Conference on Multidisciplinary Research

270

11% of bacteria cultures were not able to be

identified, and this may be attributed to the

limitations of the conventional identification system

employed.

Multidrug resistance is defined as bacteria that

are resistant to 3 or more antimicrobial classes. In

this study, 3 isolates namely Aeromonas sp., Vibrio

parahaemolyticus and Klebsiella pneumoniae were

found to be multidrug resistant (MDR). Based on

one study done by Ghaderpour et al. (2015),

emergence of resistance of bacteria is associated

with anthropogenic pollution in Matang estruary,

Kuala Sepetang. This estuary is one of the major

cockles producing area. This estuary was

contaminated with untreated silages that contain

organic materials, household chemicals and

pathogens therefore contaminated the cockles

breeding environment. The presence of these MDR

is alarming as infections caused by these resistant

organisms are difficult to treat.

5 CONCLUSIONS

This study suggests that Anadara granosa have poor

microbiological quality and harbor various

pathogenic bacteria. Trimetoprim/ sulfamethoxazole

and gentamicin are most effective in eliminating

bacteria in cockles. A number of the pathogenic

bacteria obtained namely Aeromonas sp., Vibrio

parahaemolyticus and Klebsiella pneumoniae

exhibited multidrug resistant trait.

REFERENCES

Ahmad, F., Ismail, N., Jaafar, H., Nordin, W. N., Telipot,

M., Pinang, P., & Sepetang, K., 2007. Bacteriological

Comparison Of Cockles From Three Producing Areas

In Peninsular Malaysia, 18(2), 35–44.

Akinbowale, O. L., Peng, H., & Barton, M. D., 2006.

Antimicrobial resistance in bacteria isolated from

aquaculture sources in Australia. Journal of Applied

Microbiology, 100(5), 1103–1113.

https://doi.org/10.1111/j.1365-2672.2006.02812.

Asmat, A., Mehat, D. N., Rahimi, H., & Gires, U., 2014.

Population density and antibiotic resistant of bacteria

from bivalve (Perna viridis and Anadara granosa).

Sains Malaysiana, 43(4), 543–550.

Al-Othrubi S.M.Y., Kqueen, C.Y., Mirhosseini, C.Y.,

Hadi, Y.A., Radu, S., 2014. Antibiotic Resistance of

Vibrio parahaemolyticus Isolated from Cockles and

Shrimp Sea Food Marketed in Selangor, Malaysia.

Clin Microbial 3: 148. doi:10.4172/2327-

5073.1000148

Burkhardt W, Calci K.R., 2000. Selective accumulation

may account for shellfish-associated viral illness. Appl

Environ Microbiol. 66:1375–1378. doi:

10.1128/AEM.66.4.1375-1378.2000.

Castro, D., Pujalte, M.J., Lopez-Cortes, L., Garay, E. &

Borrego, J.J. 2002. Vibrios isolated from the cultured

manila clam (Ruditapes philippinarum): Numerical

taxonomy and antibacterial activities. Journal of

Applied Microbiology 93: 438-447.

Ghaderpour, A., Ho, W. S., Chew, L.-L., Bong, C. W.,

Chong, V. C., Thong, K.-L., & Chai, L. C. (2015).

Diverse and abundant multi-drug resistant E. coli in

Matang mangrove estuaries, Malaysia. Frontiers in

Microbiology, 6, 977.

http://doi.org/10.3389/fmicb.2015.00977

Hassan R., Kanakaraju D., 2013 Razor clams (Class

Bivalvia) of Kuala Selangor, Malaysia: morphology,

genetic diversity and heavy metal concentration.

Borneo Journal of Resource Science and Technology

2(2):19–27.

Huang, C.H., Renew, J.E., Smeby, K.L., Pinkerston, K. &

Sedlak, D.L. 2001. Assessment of potential antibiotic

contaminants in water and preliminary occurrence

analysis. Water Resour. Update 120: 30-40.

Heuer, O. E., Kruse, H., Grave, K., Collignon, P.,

Karunasagar, I., & Angule, F. J., 2009. Human Health

Consequences of Use of Antimicrobial Agents in

Aquaculture. Food Safety, 1248-1253.

Iwamoto, M., Ayers, T., Mahon, B. E., & Swerdlow, D.

L., 2010. Epidemiology of seafood-associated

infections in the United States. Clinical Microbiology

Reviews, 23(2), 399–411.

https://doi.org/10.1128/CMR.00059-09

Jayasinghe, L., Ahmed, N., & Kariyawasam, U., 2005.

The Isolation and Identification of. Wayamba

University of Sri Lanka, 1–6.

Law, J. W, Ab Mutalib, N, Chan, K, & Lee, L., 2014.

Rapid methods for the detection of foodborne bacterial

pathogens: principles, applications, advantages and

limitations. Frontiers in Microbiology, 5, 770.

http://doi.org/10.3389/fmicb.2014.00770

Letchumanan, V., Chan, K.-G., & Lee, L.-H., 2014. Vibrio

parahaemolyticus: a review on the pathogenesis,

prevalence, and advance molecular identification

techniques. Frontiers in Microbiology, 5, 705.

http://doi.org/10.3389/fmicb.2014.00705

Liong, P. C., Hanafi, H. B., Merican, Z. O., Nagaraj, G.,

1988. Aquaculture development in Malaysia. In J. V.

Juario & L. V. Benitez (Eds.), Perspectives in

Aquaculture Development in Southeast Asia and

Japan: Contributions of the SEAFDEC Aquaculture

Department. Proceedings of the Seminar on

Aquaculture Development in Southeast Asia, 8-12

September 1987, Iloilo City, Philippines. (pp. 73-90).

Tigbauan, Iloilo, Philippines: SEAFDEC, Aquaculture

Department.

Ole E. Heuer, Hilde Kruse, Kari Grave, P. Collignon,

Iddya Karunasagar, Frederick J. Angulo; Human

Health Consequences of Use of Antimicrobial Agents

Occurrence of Pathogenic Bacteria in Blood Cockles, Anadara granosa

271

in Aquaculture. ClinInfect Dis 2009; 49 (8): 1248-

1253. doi: 10.1086/605667

Potasman I, Paz A and Odeh M., 2002. Infectious

outbreaks associated with bivalve shellfish

consumption: A worldwide perspective. Clin. Infect.

Dis. 35: 921–928.

Rippey, S. R., 1994. Infectious diseases associated with

molluscan shellfish consumption. Clinical

Microbiology Reviews, 7(4), 419–425.

Sartori, André F., 2015. Anadara granosa (Linnaeus,

1758). In: MolluscaBase., 2015. Accessed through:

World Register of Marine Species at

http://www.marinespecies.org/aphia.php?p=taxdetails

&id=715138 on 2017-02-01

Thompson, F.L., Iida, T. & Swings, J., 2004. Biodiversity

of Vibrios. Microbiology and Molecular Biology

Reviews 68: 403-431.

Wan Norhana N and Nor Ainy M., 2004. Bacteriological

quality of some molluscan shellfish from growing

waters of Peninsular Malaysia. Malaysia Fisheries J.

3(1): 27–38.

Yap, C. K, Razeff S. M. R, Edward F. B, and Tan S. G,

“Heavy metal concentrations (Cu, Fe, Ni and Zn) in

the clam, Glauconome virens, collected from the

northern intertidal areas of Peninsular Malaysia,”

Malaysian Applied Biology Journal, vol. 38, no. 1, pp.

29–35, 2009.

ICMR 2018 - International Conference on Multidisciplinary Research

272