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International Journal of Food Microbiology 130 (2009) 156158

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Short communication

Bacterial contaminants in carbonated soft drinks sold in Bangladesh markets

Muhammad Ali Akond a,b,, Saidul Alam a, S.M.R. Hasan a, Sanzida Mubassara a,
Sarder Nasir Uddin c, Momena Shirin d
a

Department of Botany, Jahangirnagar University, Dhaka-1342, Bangladesh

Lab of Environmental Bioscience, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh
d
Institute of Public Health, Mohakhali, Dhaka-1212, Bangladesh
b
c

a r t i c l e

i n f o

Article history:
Received 16 September 2008
Received in revised form 16 January 2009
Accepted 19 January 2009
Keywords:
Bangladesh
Carbonated soft drinks
Public health risks
Pathogenic bacteria
Coliforms

a b s t r a c t
A total of 225 carbonated soft drink (CSD) samples from nine brands, from various locations in ve
metropolitan cities of Bangladesh were examined to determine their bacteriological quality. Most samples
were not in compliance with microbiological standards set by organizations like the World Health
Organization (WHO). Pseudomonas aeruginosa was the predominant species with an incidence of 95%.
Streptococcus spp. and Bacillus stearothermophilus were the next most prevalent with numbers ranging from
6 to 122 and 9 to 105 cfu/100 ml, respectively. Fifty four percent of the samples yielded Salmonella spp. at
numbers ranging from 2 to 90 cfu/100 ml. Total coliform (TC) and faecal coliform (FC) counts were found in
68100% and 76100% of samples of individual brands, at numbers ranging from 5 to 213 and 3 to 276 cfu/
100 ml, respectively. According to WHO standards 6088% of samples from six brands and 32% and 40% of
samples from two other brands belonged to the intermediate risk group with FC counts of 1001000 cfu/
100 ml. Heterotrophic plate counts, however, were under the permissible limit in all 225 samples. These
ndings suggest that carbonated soft drinks commercially available in Bangladesh pose substantial risks to
public health.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Carbonated soft drinks (CSDs) were introduced in Bangladesh
over 35 years ago, and have since become popular in both cities and
rural areas. Since 1996 there have been no standards for, or denition
of, soft drinks in the British and EU regulations, except in special
cases (Carbonated Drinks, 2004). Protection of all types of drinks
from hazardous microbial contaminants is a global issue. Various
gastrointestinal illnesses are the most common consequences of
consuming contaminated drinks (Frobisher et al., 1974). Good
manufacturing practices (GMPs) are not always followed in the
food and beverage industries, particularly in developing countries,
and this can result in wide variations in the microbial quality of
products, such as bottled water and other drinks (Akond et al., 2006;
Hara et al., 2005; Venieri et al., 2006). Carbonated soft drinks and
bottled waters sold in Bangladesh are claimed to be of excellent
microbiological quality in their sales promotion advertisements in
both print and electronic media (Akond et al., 2006). This is in
contradiction, however, to newspaper reports of extensive algal
growth in carbonated soft drink bottles (BELA, 1997; Jaijai Din, 2008;
Janakantha, 2004), indicating that the microbiological quality of soft

Corresponding author. Lab of Environmental Bioscience, Department of Biological

Chemistry, Faculty of Agriculture, Yamaguchi University, Japan. Tel.: +81 80 3056 7275.
E-mail address: akond316@yahoo.com (M.A. Akond).
0168-1605/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2009.01.014

drinks in Bangladesh may actually be poor. Therefore, the present

study was undertaken to determine the bacterial quality of some
non-alcoholic, carbonated soft drinks available at retail outlets in
Bangladesh.
2. Materials and methods
2.1. Sampling
Intact containers (plastic and/or glass bottles or aluminum cans)
from nine brands of CSD were purchased from various retail outlets in
ve cities (Dhaka, Khulna, Rajshahi, Chittagong and Barishal) in
Bangladesh between August 2006 and March 2008. Contamination
with dust during collection and transportation was avoided by placing
containers in a sterilized foam box. The containers were washed with
sterile distilled water then sterilized by spraying and rubbing with
98% alcohol before opening. Nine brands of carbonated soft drinks
were designated arbitrarily CSD-1 to CSD-9. Five containers of each
brand from every collection site were examined.
2.2. Bacteriological analysis
A heterotrophic plate count (HPC), total coliform count (TCC),
faecal coliform count (FCC), and faecal Streptococcus, Bacillus
stearothermophilus, Pseudomonas aeruginosa, Salmonella and Shigella

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M.A. Akond et al. / International Journal of Food Microbiology 130 (2009) 156158

157

Table 1
Prevalences (%) of total coliform (TC), faecal coliform (FC) and other bacteria in groups of 25 samples of carbonated soft drinks from Bangladesh
Brand
Code

Number of positive samples

TC

FC

Salmonella

Streptococcus

Pseudomonas

Bacillus

CSD-1
CSD-2
CSD-3
CSD-4
CSD-5
CSD-6
CSD-7
CSD-8
CSD-9
Total

17 (68)
23 (92)
25 (100)
23 (92)
23 (92)
22 (88)
25 (100)
22 (88)
25 (100)
205 (91.11)

19 (76)
22 (88)
25 (100)
23 (92)
22 (88)
20 (80)
25 (100)
22 (88)
24 (96)
202 (89.78)

12 (48)
13 (52)
18 (72)
9 (36)
12 (48)
16 (64)
15 (60)
13 (52)
14 (56)
122 (54.22)

24 (96)
25 (100)
25 (100)
25 (100)
17 (68)
25 (100)
25 (100)
25 (100)
21 (84)
212 (94.22)

22 (88)
25 (100)
24 (96)
25 (100)
24 (96)
25 (100)
25 (100)
25 (100)
19 (76)
214 (95.11)

14 (56)
18 (72)
3 (12)
24 (96)
19 (76)
14 (56)
12 (48)
18 (72)
11 (44)
133 (59.11)

counts were obtained for each sample. Heterotrophic bacteria were

enumerated by spread plating 0.1 ml and pour plating a 1.0 ml
volume of each sample undiluted and in ten-fold dilutions to 10 3.
Other bacteria were enumerated by ltering 10 and 100 ml volumes
of each undiluted sample through 0.22 m membrane lters
(Millipore, Bedford, MA, USA), and incubating the lters on selective
agars. All bacteria were enumerated on triplicate plates or lters.
The culture media used for HPC, TCC, FCC, and faecal Streptococcus,
B. stearothermophilus, P. aeruginosa, Salmonella and Shigella counts
were, respectively, Nutrient agar (Difco, Becton-Dickinson, Sparks,
MD, USA), MacConkey agar (Difco), Membrane Faecal Coliform
(MFC) agar (Hi-Media, Mumbai, India), Kenner Faecal Streptococcus
(KF-Streptococcus) agar (Hi-Media), 2,3,5-triphenyl tetrazolium
chloride (TTC) agar (Sartorius, Goettingen, Germany), Cetrimid
agar (Oxoid, Basingstoke, Hampshire, UK), Bismuth Sulte agar
(Oxoid) and Salmonella-Shigella (SS) agar (Oxoid). MFC, SS and TTC
agar plates were incubated at 44 C, 42 C and 55 C, respectively,
for 24 to 72 h. All other plates were incubated at 37 C for 24 to
72 h. Characteristic colonies grown on selective and differential
agars were conrmed by the morphological and biochemical tests of
Buchanan and Gibbons (1974). The tests included gram staining,
spore staining, and tests for catalase, coagulase, oxidase, IMViC
reactions, starch hydrolysis, sugar fermentation, and nitrate
reduction.
Salmonella spp., Escherichia coli, P. aeruginosa, and Streptococcus spp.
were further conrmed by latex agglutination tests using polyvalent
antisera (DENKA SEIKEN Co. Ltd, Tokyo, Japan). Only strong agglutination occurring within 1 min was considered to be a positive reaction.
2.3. Statistical analysis
Statistical analyses of the data were carried out using SPSS 11.5 and
Microsoft Excel 2003 to calculate means, medians, standard devia-

tions, least signicant differences (LSD) and correlations, and to check

distribution normality of data sets using AndersonDarling test.
3. Results and discussion
All samples were positive for heterotrophic bacteria (Table 1). The
HPCs ranged from 3 to 242 cfu/ml (Table 2), all of which were under
the maximum permissible limit of 500 cfu/ml set by the United States
Environmental Protection Agency (US EPA, 2003). The low HPC might
be due to the incubation temperature of 37 C, as it has been claimed
that the numbers of bacteria recovered from drinks decreases with
increasing incubation temperature, from 25 to 35 C (Armas and
Sutherland, 1999; Reasoner, 2004; Rosenberg, 2003; Venieri et al.,
2006). However, a 13-fold increase in aerobic bacterial counts of
Nigerian carbonated soft drinks was obtained when culture media
were incubated at 34 C rather than 28 C (Euvweuwere and
Chynyere, 2001).
Several pathogenic and/or opportunistically pathogenic bacteria
like Salmonella, P. aeruginosa, and Streptococcus, and B. stearothermophilus were present in CSDs at numbers ranging from 2 to 90, 4 to 162,
6 to 122, and 9 to 105 cfu/100 ml, respectively (Table 2). Salmonella
enteritidis, S. typhimurium, E. coli, Pseudomonas spp. and Streptococcus spp. were conrmed by polyvalent antisera agglutination tests.
The ranges for TC and FC counts were 5 to 213 and 3 to 276 cfu/
100 ml, respectively. Ninety percent of samples were non-compliant
with the guidelines of the World Health Organization (WHO, 1997);
brands CSD-3 and CSD-7 were also 100% non-compliant with
guideline values for faecal coliform (Table 3). Sixty ve percent of
the samples were in the intermediate risk group, while 4% of samples
were in the high risk group. Such high counts were found by Kohnen
et al. (2005) for both coliform and Pseudomonas spp. in 39 and 12% of
samples of home-prepared carbonated water, respectively, as compared to only 12 and 4%, respectively, of samples of tap water used for

Table 2
Medians (ranges) of numbers of bacteria recovered from groups of 25 samples of nine carbonated soft drinks from Bangladesh
Brand
code
CSD-1
CSD-2
CSD-3
CSD-4
CSD-5
CSD-6
CSD-7
CSD-8
CSD-9
a
b
c
d

Median and range for bacterial counts (cfu/100 ml)

HPCa
0.8 (0.153.25)
12.2 (2.923)
3.2 (1.610)d
7.2 (2.224)d
4 (2.38)
1.4 (0.43.4)d
7.5 (410)
8 (124.2)d
7 (117)

TCb
d

9.0 (046)
27 (0213)d
27 (948)
28 (053)
35 (071)
16 (051)d
32 (8146)d
56 (0110)
32 (990)d

Heterotrophic plate count, cfu 103/100 ml.

Total coliform.
Faecal coliform.
Data set not normally distributed.

FCc

Salmonella

Streptococcus

Pseudomonas

Bacillus

8 (022)d
20 (037)
20 (6276)d
20 (050)
15 (045)
9 (049)d
21 (546)
34 (092)
45 (0160)d

0 (058)d
13 (087) d
12 (052) d
0 (017)d
0 (045)d
8 (027)d
8 (069)d
2 (020)d
12 (090)d

14 (033)
88 (12122)d
27 (960)d
19 (651)
8 (029)d
13 (640)d
33 (883)
61 (1283)
11 (019)

22 (058)
81 (28128)
20 (043)
33 (1266)
27 (061)
29 (959)
38 (9107)
132 (54162)d
12 (036)

9 (023)d
22 (062)
0 (012)d
51 (0105)
12 (045)
9 (025)d
0 (033)d
21 (058)
0 (022)d

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Table 3
Numbers of samples (%), in groups of 25, positive for World Health Organization risk
groups based on faecal coliform (FC) counts
Brand
code

Risk category
Nonea

Lowb

Intermediatec

Highd

CSD-1
CSD-2
CSD-3
CSD-4
CSD-5
CSD-6
CSD-7
CSD-8
CSD-9
Total

6 (24)
3 (12)
0 (0.0)
2 (8.0)
3 (12)
5 (20)
0 (0.0)
3 (12.0)
1 (4.0)
23 (10.22)

11
2
4
1
5
10
6
3
4
46

8 (32)
20 (8)
16 (64)
22 (88)
17 (68)
10 (40)
19 (76)
19 (76)
15 (60)
146 (64.89)

0
0
5 (20)
0
0
0
0
0
5 (20)
10 (4.44)

a
b
c
d

(44)
(8.0)
(16)
(4.0)
(20)
(40)
(24)
(12)
(16)
(20.44)

FC = 0.
FC = 110 cfu/100 ml.
FC = 10100 cfu/100 ml.
FC = 1001000 cfu/100 ml.

be causes for the high counts. Extensive algal growth and the presence
of sediments in bottles of carbonated soft drinks have been reported in
Bangladeshi newspapers (BELA, 1997; Jaijai Din, 2008; Janakantha,
2004), which also indicates the poor quality of carbonated soft drinks.
The presence of such materials and the poor microbiological quality
reported in the current study are the result of failure to maintain the
acceptable quality and to adequately maintain the regulations of
Bangladesh Government in the manufacture of carbonated soft drinks.
Immediate action to improve the microbiological quality of carbonated beverages in Bangladesh is evidently required.
Acknowledgement
Authors are thankful to the Lab of Microbiology, Department of
Botany, Jahangirnagar University, and Bacteriology Laboratory,
Institute of Public Health, Dhaka, Bangladesh.
References

their preparation. The high rate of contamination in the carbonated

waters was likely due to biolms in the water bottles. According to
WHO (1997) and the International Commission on Microbiological
Specications for Foods (1986), bottled water should be produced
without contamination by coliforms and most Pseudomonas.
LSD analysis showed signicant differences between the mean
counts of HPC, TC and FC in most samples (p = 0.01). There was no
correlation between the numbers of any two groups of organisms
(r = 0.47), except for weak correlations between TC and FC for brands
CSD-8 (r = 0.66) and CSD-5 (r = 0.58) and between B. stearothermophilus and P. aeruginosa for brand CSD-5 (r = 0.59).
The fractions of the 225 samples positive for other bacteria ranged
from 54% for Salmonella spp. to 95% for P. aeruginosa (Table 1). In a
Canadian study, Warburton et al. (1998) reported b1 cfu/ml of Pseudomonas spp. in carbonated bottled mineral water in all samples
tested. Though Pseudomonas is frequently noticed in bottled water
and drinking water (Rosenberg, 2003), it is not normally a health
hazard, however, it can cause infections to immunocompromised
individuals. Reports of Salmonella spp. in bottled water or carbonated
beverages are uncommon. The high counts of Salmonella spp. in
carbonated soft drinks in Bangladesh are obviously a risk for public
health. The presence of Salmonella spp. is an indication of enteric
contamination and may have been introduced to these soft drinks
during bottling and/or manufacturing, due to lack of personal hygiene.
Of the total samples in the current study, 59% were positive for B.
stearothermophilus. This high rate of Bacillus spp. was also found in
Cola samples from Nigeria, with incidence of up to 81%. Given the
environmental occurrence, as well as the osmotic, thermal and salt
stress tolerance of the bacteria, Bacillus spp. could be introduced in
these drinks from either or both the environment and/or from raw
materials (Euvweuwere and Chynyere, 2001; Oranusi et al., 1994).
The presence of B. stearothermophilus, which is used as an indicator in
sterilization validation tests, indicates improper treatment of water
used in the production of these drinks. This is in negligence of the
regulations of the Bangladesh Government, which states that the
water to be used in the production of carbonated soft drinks must be
drawn from an underground depth of at least 300 ft, it must not
contain heterotrophic bacteria at more than 50 cfu/ml, and must be
negative for all TC and FC counts (BSTI, 2001).
The presence of high numbers of TC, FC and various harmful
bacteria, especially Salmonella spp., in carbonated soft drinks in
Bangladesh poses a serious public health risk. Poor manufacturing
processes, and ingredient and/or water source contamination may all

Akond, M.A., Alam, S., Shil, A., Hasan, S.M.R., 2006. Bacteriological quality of bottled
water available commercially in Bangladesh. Journal of Environmental Sciences
(Dhaka) 4, 4752.
Armas, A.B., Sutherland, J.P., 1999. A survey of the microbiological quality of bottled
water sold in the UK and changes occurring during storage. International Journal of
Food Microbiology 48, 5965.
BELA, 1997. PIL (List of Public Interest Litigation), No. 11, Dr. Mohiuddin Farooque v.
Bangladesh & others, Writ Petition No. 867/97 (Contaminated Drink). Bangladesh
Environmental Lawyers Association (BELA), Segunbagicha, Dhaka. http://www.
belabangla.org/html/pil.htm. Accessed on December 30, 2008.
BSTI, 2001. Standard for Bottled Drinking Water/ Soft Drinks. BDS:1240, 2001.
Bangladesh Standard Testing Institute (BSTI), Dhaka.
Buchanan, R.E., Gibbons, N.E. (Eds.), 1974. Bergey's Manual of Determinative Bacteriology,
8th ed. Williams and Willkins Co., Baltimore.
Carbonated Drinks, 2004. British Soft Drinks Association, 2022 Stukeley Street,
London. http://www.liquidsmeanlife.org.uk/member/KS3&4%20new%20material/
Teachers%20notes/Carbonated%20Drinks.pdf. Accessed on December 15, 2008.
Daily Jaijai Din, 2008. Soft Drinks Shorirer Jonno Soft Noy (Soft Drinks are not soft for
human body), February 14, 2008. Jaijai Din Publications Ltd., Tejgaon, Dhaka,
Bangladesh, p. 16.
Daily Janakantha, 2004. Coker Botole Shaola (Algae in bottle of Coke), March 23, 2004.
Janakantha Ltd., Janakantha Bhaban, New Eskaton Road, Dhaka, Bangladesh, p. 1.
Euvweuwere, B.J.O., Chynyere, M., 2001. The microbiology and deterioration of soft
drinks subjected to two different marketing. Global Journal of Pure and Applied
Sciences 7, 4348.
Frobisher, M., Hinsdill, R.D., Crabtree, K.T., Goodheart, C.R., 1974. Fundamentals of
Microbiology, 9th ed. W.B. Saunders Company, Philadelphia.
Hara, M., Fukuyama, M., Furuhata, K., 2005. Isolation of thermotolerant acidophilic
bacteria (TAB) from fruit juices. Journal of Antibacterial and Antifungal Agents 9,
447452.
International Commission on Microbiological Specications for Foods, 1986. Microorganisms in foods, 2, Sampling Plants for Natural Mineral Waters, other bottled
waters, Process Waters and Ice, 2nd ed. Blackwell Scientic Publications, London.
Kohnen, W., Teske-Keiser, S., Meyer, H.-G., Loos, A.H., Pietsch, M., Jansen, B., 2005.
Microbiological quality of carbonated drinking water produced with in-home
carbonation system. International Journal of Hygiene and Environmental Health
208, 415423.
Oranusi, S.U., Ezeogu, L.I., Okolo, B.N., 1994. Microbial contaminants of commercially
bottled non-alcoholic drinks produced in Nigeria. World Journal of Microbiology
and Biotechnology 10, 488490.
Reasoner, D.J., 2004. Heterotrophic plate count methodology in the United States.
International Journal of Food Microbiology 92, 307315.
Rosenberg, F.A., 2003. The microbiology of bottled water. Clinical Microbiology
Newsletter 25, 4144.
US EPA, 2003. List of drinking water contaminants and maximum contaminant levels,
USEPA. Ofce of GWDW, EPA, Washington, DC. Publication No. 816-F-03-016.
Venieri, D., Vantarakis, A., Komninou, G., Papapetropoulou, M., 2006. Microbiological
evaluation of bottled non-carbonated (still) water from domestic brands in
Greece. International Journal of Food Microbiology 107, 6872.
Warburton, D., Harrison, B., Crawford, C., Foster, R., Fox, C., Gour, L., Krol, P., 1998. A
further review of the microbiological quality of bottled water sold in Canada: 1992
1997 survey results. International Journal of Food Microbiology 39, 221226.
WHO, 1997. 2nd ed. Guideline for Drinking Water Quality, vol. 3. World Health
Organization, Geneva, Switzerland.