Pergamon Environment International, Vol. 25, No. 1, pp.

53-57, 1999
Copyright © 1999 Elsevier Science Ltd
Printed in the USA. All rights reserved
0160-4120/99/S-see front mauex

PII S0160-4120(98)00097-X

MICROBIOLOGICAL QUALITY OF BOTTLED

DRINKING WATER IN THE UAE AND THE EFFECT
OF STORAGE AT DIFFERENT TEMPERATURES

H. Nsanze and Z. Babarinde

Faculty of Medicine and Health Sciences, P.O. Box 17666, AI Ain, United Arab Emirates
e-mail: nsanze@emirates.net.ae

H. AI Kohaly
Department of Microbiology, AI Ain Hospital, United Arab Emirates University, United Arab
Emirates

E1 9803-131 M (Received 23 March 1998; accepted 5 September 1998)

The microbiological quality of bottled water from different sources in the United Arab Emirates
was studied. The study was done on 80 commercial bottled water samples from 4 different
manufacturing companies. The results showed that 75% of the 20-L bottles were contaminatedby
10 different species of bacteria, whereas 10 to 40% of the 1.5-L bottles were contaminated by 2-4
types of micro-organisms. Heterotrophic bacteria and a few types of human-associated bacteria
were isolated. The most frequent organism found in all types of water was Acinetobacter lwoffii
with several genospecies. Storage of water at 4°C preserved the water without microbial
multiplication, whereas at 25-37°C, most microbes multiplied, and at 42°C, most contaminants
were destroyed. The source of these micro-organisms and their untoward effects on the drinking
water were not determined. ©1999ElsevierScienceLtd

INTRODUCTION

The quality o f bottled drinking water is variable in
many parts o f the world. There are many factors that
influence the quality of this type o f water. The drinking
habits are also diverse and depend on environmental
and economic factors. Bottled mineral drinking water
is extensively marketed in the United Arab Emirates
(UAE) and the whole Arabian peninsula. Plenty of
bottled water is consumed daily by the population
because this region is mainly a desert with high temp-
eratures, low humidity, and no free-flowing water.
There is a significant level of consumption o f bottled
mineral water in the United States and Canada; in the
province o f Qu6bec with a population of 6 800 000,
about 13% now drink bottled water (MEQ 1991). In the
UAE, however, nearly 90% of the population drink
bottled mineral water.
The water quality is often related to the degree of
bacterial contamination. The quantity of bacteria in
commercial mineral water is generally dependent on
the disinfecting process of natural spring water used at
the factory. It is well known that natural mineral water
is characterized by its bacterial flora, chemical and
physical composition. The quantity o f microbial flora
of spring water is usually high. Spring water contains
a natural microflora composed mainly of species o f the
genera Achromobacter, Flavobacterium, Alcaligenes,

53
54
Acinetobacter, Cytophaga, Moraxella, and Pseudo-
monas (Gonzales et al. 1987; Kolbel-Boelke et al.
1988). If these microorganisms are not adequately
removed during processing and bottling, they will be
found in commercial drinking water.
Additional microbial contamination can occur at any
time during and after processing. In Wales, Hunter and
Burge (1987), found Staphylococcus epidermidis and
Staphylococcus hominis in 6 of 52 bottles of water,
which was attributed to poor hygiene during pro-
cessing. In Canada, 2 to 3% of the samples contained
coliform bacteria. Sekla (1988) found 2 out of 60 bott-
les of water from retail stores and processing plants to
contain coagulase-positive Staphylococcus aureus,
Enterococcus spp. in 1 sample and coliforms in 4 sam-
ples.
Although the microbial quantity in processed water
is initially small, it can evolve rapidly to high numbers
during storage (Stickler 1992). In the absence of treat-
ment with chlorination or ozonation, bacterial multi-
plication may occur for 1 to 3 weeks after bottling, and
the bacterial count can reach 103 to 104 bacteria per mL
at 37°C (Gonzales et al. 1987). It is known that storage
at ambient temperatures will aid the multiplication of
contaminants contained therein. Water in this region is
usually stored at any temperature between 4 and 42 °C.
The Acinetobacter species, Pseudomonas species,
and Stenotrophomonas maltophilia are opportunistic
pathogens to immunocompromise. The presence of
these organisms acts as an indicator of the possible pre-
sence of pathogenic agents, including Cryptosporidium
parvum, which is commonly found in water (Goldstein
et al. 1996). Outbreaks of waterborne diseases asso-
ciated with untreated drinking water are common (Gale
1996; Kramer et al. 1996), but diseases from bottled
spring water are uncommon. Recently an outbreak of
non-O 1- Vibrio cholerae involving 11 people was
reported associated with large reusable bottles in the
United States (CDC 1996). During that outbreak,
bottled water tested positive for coliforms but the
source of contamination was not determined. Some
organisms, such as Pseudomonas aeruginosa, have a
synergistic effect on the survival of pathogenic organ-
isms like salmonellae, enabling them to survive for
more than 140 d in double-distilled water (Warburton
et al. 1994).
Another contributing factor to the degree of conta-
mination is the ease with which bacteria multiply in
polyvinyl chloride (PVC) containers. When water is
heavily contaminated, it gives off a bad odor and may
H. Nsanze et al.
cause gastrointestinal upset. It is known that PVC
bottles used, in association with certain bacteria, such
as Acinetobacter species and Stenotrophomonas
maltophilia, frequently have an off-odor, due to the
phenomenon of elevated lipolytic activity due to their
cell hydrophobicity and adhesivity to the PVC. These
bacteria attack the sodium polysulfide included in the
ultramarine blue dye present in PVC, transforming it to
hydrogen sulphide (Guerzoni et al. 1994; Moreira et al.
1994).
It appears that there are no published data on the
bacteriological quality of the various bottled waters
consumed in the UAE and the effects of storage or the
methods of minimizing contaminants. The aim of this
study was to determine if contamination of bottled
mineral water occurred at the time of delivery from the
factory to the consumer or retail shops, and to advise
on better methods of contamination control.
MATERIALS AND METHODS
The water studied for the rate of contamination in-
eluded samples taken from 3 types of commonly con-
sumed 1.5-L bottles of water (labeled as manufacturers
"M", "A", and "G") purchased from a supermarket
close to the laboratory. The fourth water type investi-
gated was from 20-L bottles (labeled as manufacturer
"O"), delivered weekly to the laboratory within 2 d of
bottling. Twenty bottles of each type of water were
sampled and analyzed in a period of three months.
The second part of the study involved only water
drawn from 10 "O" type bottles. The study was con-
ducted on 10 different delivery batches. Each bottle
was sampled on opening then portioned into 4 aliquots
which were stored at 4 °, 25 °, 37 °, and 42°C. Each
aliquot was sampled and analyzed daily for bacterial
counts over the next three consecutive days.
The bacterial counts as colony forming units (CFU)
were determined by a surface pour plate technique.
Each bottle was aseptically opened and a sample of
10 mL of water poured into a sterile plastic tube. Then
100 lxL of water was placed on a CLED medium on a
9 mm petri dish and spread evenly over the entire
surface. The plates were incubated at 37°C for 5 d,
after which a total bacterial count was performed.
Initially this method was compared to a micropore
filter membrane technique and found comparable and
accurate. The isolate organisms were identified as far
as possible mainly using API 20E, API 20NE kits
(BioMerieux) and other necessary laboratory methods.
Microbiological quality of UAE bottled drinking water 55

Table 1. Rate of bacterial contamination of bottled drinking water.

Water volume Percent

Manufacturer L No. of bottles No. growing contaminated
(M) 1.5 20 2 10.0
(A) 1.5 20 5 25.0
(G) 1.5 20 8 40.0
(O) 20.0 20 15 75.0

Table 2. Bacterial isolates from bottled drinking water.

Organisms isolated <100
Manufacturer "M"
Acinetobacter lwoffii
Unidentified GPB
Manufacturer "A"
Acinetobacter lwoffii
Bacillus species
Micrococcus species
Manufacturer "G"
Acinetobacter lwoffii
Acinetobacter spp
Steno. maltophilia
Unidentified GPB
Manufacturer "O"
Acinetobacter lwoffii
Acinetobacter spp
Pseudomonas species
Steno. maltophilia
Flav. meningosepticum
Erwinia spp
Turtumella pteos
Edwardsiella iictaluri
Staph. epidermidis
Unidentified GPB
GPB = Gam positive bacilli.
CFU / mL
100-1000 >1000
Number of bottles with growth
1 0
0 1
0 2
1 0
1 0
0 2
0 0
1 0
0 1
2 2
4 2
4 1
1 1
1 0
1 0
1 0
1 0
1 0
1 0
Total no.
of bottles
0 1
0 1
1 3
0 1
0 1
3 5
1 1
0 1
0 1
3 7
I 7
1 6
1 3
0 1
0 1
0 I
0 1
0 1
1 2

RESULTS nation rate o f the smaller, 1.5-L bottles, w a s lower at

In a typical g r o w t h o f m i x e d o r g a n i s m s f r o m a
100#tL s a m p l e f r o m m a n u f a c t u r e r " O " there w e r e
a b o u t 8 different colonial m o r p h o t y p e s . Table 1 shows
the rate o f c o n t a m i n a t i o n for the 4 different types o f
bottles. Fifteen ( 7 5 % ) o f the 2 0 - L bottles (type O) w e r e
c o n t a m i n a t e d b y 10 different bacteria. The c o n t a m i -
10% in type M, 2 5 % in type A, and 4 0 % in t y p e G.
Table 2 shows the quantity, n u m b e r o f bottles (out o f
20) contaminated, and the t y p e o f c o n t a m i n a n t s in the
4 different m a n u f a c t u r e r s ' bottles. T h e r e w e r e 2 types
o f m i c r o - o r g a n i s m s in type " O " , including Acineto-
bacter lwoffii in small quantities. T h e r e w e r e 3 and 4
different m i c r o - o r g a n i s m s in types " M " and " A " ,
56 H. Nsanze et al.

Table 3. Bacterial count for water stored at different temperatures for 3 d.

Sample number
Temperature and duration 1 2 3 4 5 6 7 8 9 10
Bacterial counts (CFU)
Start 74 112 200 800 240 0 36 0 5 0

4°C .. 24 h 75 72 400 800 100 0 20 0 14 0

4°C .. 48 h 70 79 750 500 50 0 15 0 6 0
4°C .. 72 h 80 102 >1000 >1000 300 0 52 0 3 0

25°C .. 24 h 75 72 >1000 >1000 160 0 1500 0 300 0

25°C .. 48 h 87 400 >1000 >1000 300 4 1500 12 72 0
25°C .. 72 h 86 >1000 >1000 >1000 400 500 1500 31 300 0

37°C .. 24 h >1000 250 300 500 80 0 1400 0 410 0

37°C .. 48 h >1000 300 300 600 20 500 1400 2 300 0
37°C .. 72 h >1000 500 40 >1000 4 500 900 2 42 0

42°C .. 24 h 0 0 0 30 5 0 220 0 0 0
42°C .. 48 h 0 0 0 46 300 2 220 0 0 0
42°C .. 72 h 0 0 0 40 4 1 120 0 0 0

respectively. In these bottles, there w a s an increase in
the quantity and f r e q u e n c y o f Acinetobacter lwoffii.
In type " A " , there was an additional Acinetobacter
species and Stenophormonas maltophilia. There w e r e
10 different m i c r o - o r g a n i s m s found in 15 o f the 20-L
bottles. Acinetobacter lwoffii and Acinetobacter
species w e r e found in 12 o f the bottles. The counts o f
Acinetobacter w e r e m u c h higher than in the 1.5-L
bottles. C o l i f o r m s belonging to Erwinia species,
Turtumella pteos, and Edwardsiella iictaluri were
f o u n d on 3 occasions in 2 bottles. Staphylococcus
epidermidis w a s isolated f r o m this type o f bottle only.
T a b l e 3 shows the survival and multiplication o f
c o n t a m i n a n t s at different t e m p e r a t u r e s for a period o f
3 d only. There was only one o f the 10 s a m p l e d bottles
that initially contained no detectable bacteria and
r e m a i n e d the s a m e throughout the storage period at all
temperatures. T w o other bottles had no detectable or-
g a n i s m s at the beginning, but had growth at 25 ° , 37 ° ,
and 4 2 ° C , one o f t h e m having no growth at 42°C. The
r e m a i n i n g 7 bottles with initial contamination showed
no change at 4 ° C in 4 samples but s o m e increase in 2
s a m p l e s at 72 h. At 2 5 ° C , 4 o f the 7 samples showed
no change at 24 h, while 3 increased significantly. This
change w a s m a i n t a i n e d up to 72 h. At 37°C, 5 o f the
7 initially c o n t a m i n a t e d had significantly increased
counts b y 24 h but, in 3, the count declined o v e r the
3 d. At 42°C, 4 o f the 7 c o n t a m i n a t e d had lost all the
contaminants within 24 h and this w a s maintained
throughout. The bacterial count in the other 3 samples
tended to diminish.
DISCUSSION
The bacterial findings o f the investigated bottles
showed that there w a s a g o o d proportion o f conta-
mination o f each type o f bottle. T h e p r e v a l e n c e and
quantity o f contamination varied according to the type
o f bottle. Although none o f the bottled drinking water
investigated had strict pathogenic bacteria, a large
n u m b e r o f bottles contained heterophilic bacteria.
Similar and excessive contamination has been reported
f r o m Europe ( K o l b e l - B o e l k e et al. 1988) but not f r o m
N o r t h A m e r i c a n studies (Warburton et al. 1992).
The rate and frequency o f microbial contamination in
the larger 2 0 - L bottles was higher than the 1.5-L
bottles. There are several factors responsible for the
difference but a notable factor w a s that these larger 20-
L bottles were reusable, and are difficult to clean with
no possibility o f sterilization. Bacteria, protozoa, and
fungi have often been o b s e r v e d in long-standing resi-
dual w a t e r in s o m e o f these bottles. The difference in
the quality and quantity o f c o n t a m i n a n t s b e t w e e n the
smaller 1.5-L bottles is due to different spring water
Microbiological quality of UAE bottled drinking water
sources and v a r y i n g degrees o f efficiency o f the m a n u -
facturers for the w a t e r disinfection process.
A s bottled drinking w a t e r in the U A E is n o r m a l l y
stored at a t e m p e r a t u r e v a r y i n g b e t w e e n 4 and 45°C,
the c o n t a m i n a t i o n will be aided b y temperature. This
study d e m o n s t r a t e d that the o r g a n i s m s multiply m o r e
easily b e t w e e n 2 5 ° C and 37°C, the c o m m o n indoor
temperature, w h e r e a s refrigeration or high t e m p e r a -
tures (around 4 2 ° C ) reduce or maintain the status quo
for at least 3 d. It is thus unfortunate that the o p t i m u m
t e m p e r a t u r e for multiplication is the n o r m a l r o o m
t e m p e r a t u r e in U A E air-conditioned houses.
It is not possible, at the m o m e n t , to place any public
heath significance on these bacterial isolates in the
drinking water. T h e s e m i c r o - o r g a n i s m s are usually
n o n p a t h o g e n i c for h u m a n s , except for i m m u n o c o m p r o -
m i s e d patients. H o w e v e r , s o m e m e m b e r s o f the c o m -
m o n l y isolated agents, such as Acinetobacter species
and Stenotrophomonas maltophilia, h a v e recently been
subject to research b e c a u s e they are ubiquitous organ-
isms and are gaining increasing medical importance.
CONCLUSION
All f o u r t y p e s o f bottled mineral w a t e r investigated
in the U A E contained s o m e bacterial contamination.
There w e r e m o r e than 10 different species o f contami-
nating bacterial agents. Acinetobacter lwoffii was the
m o s t frequent isolate. The n o r m a l storage t e m p e r a t u r e
w a s a contributing factor in the multiplication o f con-
taminants. T h e degree and rate o f c o n t a m i n a t i o n sug-
gest a need to be cautious and vigilant to avert the
possibility o f u n t o w a r d effects o f these types o f drink-
ing water.
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