The Microbiological Quality of Ice Used to Cool Drinks and Foods in Ogbomoso Metropolis, Southwest, Nigeria

Abstract

The microbiological safety of commercial ice used to refrigerate drinks and fish was evaluated using 40 ice samples collected from four ice manufacturing factories in Ogbomoso, Nigeria. All the samples were contaminated by bacteria and the microbial load ranged from 1.88 to 3.20 × 10 4 cfu/ml which is largely above the recommended loads of <500 and <1000 cfu/ml for ice obtained from manufacturing plants and retail outlets, respectively. The bacterial isolates obtained from the ice samples include Pediococcus cerevisiae, Bacillus subtilis, Streptococcus pyogenes, Bacillus firmus, Pseudomonas aeruginosa, Streptococcus equi, Staphylococcus epidermidis, and Micrococcus luteus. The degree of resistance shown by the isolates to the antibiotics differs ranging from 50-87.5%, with multiple-drug resistance to 4-7 antibiotics. The isolates showed 100% resistance to Cotrimoxazole, Ampicillin, Cefotaxine and Cephalexin, while none of them was resistant to Gentamicin. The resistance to other antibiotics ranged from 26.67% for Ofloxacin, 66.67% for Erythromycin to 86.67% for Tetracycline. The present study reveals that ice may represent novel route of spread of antibiotic– resistant bacteria especially in developing countries. In view of the results herein reported, it is highly recommended that national regulatory guidelines should be established for the production of ice. Commercial ice should be safe to consume and be of the same quality as drinking water because it is ingested directly when added to juices and soft drinks or indirectly when used to refrigerate foods such as fish and seafoods (Falcão et al., 2002). Ice is sometimes contaminated with pathogenic microorganisms where a contaminated water source is used in its production or where there are lapses in hygiene in their handling. Outbreaks of gastroenteritis due to contaminated ice have been reported (Quick et al., 1992; Khan et al., 1994; Pedalino et al., 2003) in other parts of the world. The possible causes of these outbreaks were due to the consumption of ice contaminated with pathogens such as Norovirus and Giardia lamblia. An investigation revealed that a server's hands might have contaminated ice machines with Norovirus and there was direct transfer from the hands of a Giardia lamblia carrier who scooped up ice for restaurant customers with her contaminated bare hands (Quick et al., 1992). Recently, a major outbreak of hepatitis A in Lampang and Chiang Rai, Thailand, affecting about nine hundred people, was also reportedly due to contaminated ice (APEC, 2005). Initial investigations pointed to an ice factory in Chiang Rai Province which drew its water from contaminated artesian wells.

Full text

The Microbiological Quality of Ice Used to Cool Drinks and Foods in
Ogbomoso Metropolis, Southwest, Nigeria
Agbaje Lateef
1
*, Julius K. Oloke
1
, Evariste B. Gueguim Kana
1
, and Esther Pacheco
2
1
Departments of Pure and Applied Biology, Ladoke Akintola University of Technology,
PMB 4000, Ogbomoso, Nigeria,
2
Science Laboratory Technology,
Ladoke Akintola University of Technology, PMB 4000, Ogbomoso, Nigeria.
The microbiological safety of commercial ice used to refrigerate drinks and fish was evaluated using 40
ice samples collected from four ice manufacturing factories in Ogbomoso, Nigeria. All the samples were
contaminated by bacteria and the microbial load ranged from 1.88 to 3.20 × 10
4
cfu/ml which is largely above
the recommended loads of <500 and <1000 cfu/ml for ice obtained from manufacturing plants and retail outlets,
respectively. The bacterial isolates obtained from the ice samples include Pediococcus cerevisiae, Bacillus
subtilis, Streptococcus pyogenes, Bacillus firmus, Pseudomonas aeruginosa, Streptococcus equi,
Staphylococcus epidermidis, and Micrococcus luteus. The degree of resistance shown by the isolates to the
antibiotics differs ranging from 50-87.5%, with multiple-drug resistance to 4-7 antibiotics. The isolates showed
100% resistance to Cotrimoxazole, Ampicillin, Cefotaxine and Cephalexin, while none of them was resistant to
Gentamicin. The resistance to other antibiotics ranged from 26.67% for Ofloxacin, 66.67% for Erythromycin to
86.67% for Tetracycline. The present study reveals that ice may represent novel route of spread of antibiotic–
resistant bacteria especially in developing countries. In view of the results herein reported, it is highly
recommended that national regulatory guidelines should be established for the production of ice.
Commercial ice should be safe to consume and be of
the same quality as drinking water because it is ingested
directly when added to juices and soft drinks or indirectly
when used to refrigerate foods such as fish and seafoods
(Falcão et al., 2002). Ice is sometimes contaminated with
pathogenic microorganisms where a contaminated water
source is used in its production or where there are lapses in
hygiene in their handling. Outbreaks of gastroenteritis due
to contaminated ice have been reported (Quick et al., 1992;
Khan et al., 1994; Pedalino et al., 2003) in other parts of the
world. The possible causes of these outbreaks were due to
the consumption of ice contaminated with pathogens such
as Norovirus and Giardia lamblia. An investigation
revealed that a server’s hands might have contaminated ice
machines with Norovirus and there was direct transfer from
the hands of a Giardia lamblia carrier who scooped up ice
for restaurant customers with her contaminated bare hands
(Quick et al., 1992). Recently, a major outbreak of hepatitis
A in Lampang and Chiang Rai, Thailand, affecting about
nine hundred people, was also reportedly due to
contaminated ice (APEC, 2005). Initial investigations
pointed to an ice factory in Chiang Rai Province which
drew its water from contaminated artesian wells.
*Corresponding author, mailing address: Department of Pure and
Applied Biology, Ladoke Akintola University of Technology,
PMB 4000, Ogbomoso, Nigeria. Phone: +234-8037400520. E-
mail: agbaje72@yahoo.com
A number of studies from different countries have shown
that the microbiological quality of ice manufactured for use
to cool foods and drinks could be a cause for concern
(Moyer et al., 1993; Wilson et al., 1997; Vieira et al., 1997;
Nichols et al., 2000). These studies showed that E. coli,
coliforms and a variety of microorganisms could be present
in ice demonstrating either the poor quality of source water
used or a lack of hygiene in production or handling or both.
If the quality of source water is not good, harmful
microorganisms may be present and since the process of
freezing cannot destroy them, many of them can survive in
ice, although their numbers reduce gradually with time.
Although when ice is thawed the microorganisms remaining
may be injured, but they tend to recover their viability so
that when the ice melts into drinks, they may be able to
survive there too (FEHD, 2005). This means that if harmful
microorganisms are present in the source water from which
the ice is made, they may also be viable in the ice when it is
used, and capable of causing infection in the customer.
Therefore the relationship between contaminated water and
human diseases emphasizes the importance of a study to
gain information about the hygienic conditions of
commercial ice. In Nigeria, there is dearth of information
on the microbiological contamination of commercial ice
used to refrigerate drinks and foods, and the attendant
public health implications.
Internet Journal of Food Safety, Vol. 8, 2006, p. 39-43
Copyright© 2004, Food Safety Information Publishing
39

The purpose of the present study is to determine the
microbiological quality of edible ice from ice
manufacturing plants in Ogbomoso metropolis, Southwest
Nigeria. The bacterial isolates were evaluated to determine
their resistances to the commonly recommended antibiotics
in Nigeria. The results will provide scientific information to
assess the risk of edible ice to public health in a developing
nation such as Nigeria, and assist in setting guidelines for
the hygienic production of ice as it is presently obtainable
in the production of sachet water in the country.
MATERIALS AND METHODS
Sampling. A total of 40 ice samples were aseptically
collected from four ice manufacturing factories in
Ogbomoso, Oyo state, Nigeria. The ice samples were
prepared from water obtained from deep wells. The
sampling period was between August and November, 2005.
The samples were kept below 5
o
C during transportation to
the laboratory and were analyzed within 4h of collection.
Microbiological analysis. The samples were allowed to
melt at 5
o
C under aseptic condition, after which they were
used for the analysis. The total colony count was done by
pour plate method using nutrient agar (Lateef, 2004; Lateef
et al., 2005). The ice water samples were serially diluted
using sterile distilled water, and 0.2 ml of appropriate
dilution was used to inoculate the plate in duplicate. The
plates were incubated at 37
o
C for 24 h, after which the total
colony count was determined. Distinct colonies based on
colonial morphology were purified to obtain pure cultures
that were subjected to routine primary and biochemical tests.
The isolates were identified according to the scheme of
Buchanan and Gibbons (1974). The three-tube procedure
using lactose broth (Hammad and Dirar, 1982; Fawole et al,
2002; Bakare et al., 2003) was used to detect the coliform
and determine the most probable number (MPN) of
coliform bacilli using McCrady table. A 0.1 ml, 1 ml, and
10 ml of each sample were used to inoculate the lactose
broth in five replicates. Tubes were incubated at 37
o
C for
48 h and the MPN was determined in accordance with
standard method (APHA, 1985). For the detection of fecal
coliform bacteria, production of acid and gas was taken as
positive indication (D’Auriac et al., 2000).
Antibiotic sensitivity test. The bacterial isolates were
tested for their sensitivity to antibiotics by means of M2-A6
disc diffusion method recommended by the National
Committee for Clinical Laboratory Standards, NCCLS
(NCCLS 1997) using nutrient agar. The commercial discs
used contained the following: Gentamicin (Gen) 10 µg;
Tetracycline (Tet), 30 µg; Cotrimoxazole (Cot), 25 µg;
Nalidix acid (Nal), 30 µg; Ampicillin (Amp), 25 µg;
Cefotaxine (Cro), 30 µg; Ofloxacin (Ofl), 10 µg;
Cephalexin (Crl), 30 µg; and Erythromycin (Ery), 5 µg. The
plates were incubated at 37
o
C for 24 h, after which the
zones of inhibition were examined and interpreted
accordingly (Chortyk et al., 1993) considering the
appropriate breakpoints (Andrews, 2005). Earlier, the
potencies of all the antibiotics used in the study were
confirmed using susceptible E. coli strains.
RESULTS
The attributes of the ice samples depicting the source,
type, use, the microbial load of heterotrophic bacteria and
the bacterial isolates are as shown in Table 1. The ice
samples were produced by the factories for onward use by
consumers in different forms. They are used for diverse
purposes, which include cooling of water and drinks that
are directly consumed by humans without further treatment.
Also, a sizeable amount of the ice is used to refrigerate fish.
The water samples used to produce these ice samples were
obtained from deep wells. In Nigeria, the National agency
for food and drug administration and Control (NAFDAC) in
its guidelines listed well and deep well water as
unacceptable sources of water for the production of package
water (NAFDAC, 2004). This is because in most Nigerian
cities, the general mode of disposal of sewage is by the use
of the cesspools, septic tanks and pit latrines. And except
for very few factories now, there are no sewer and modern
sewage treatment plants. Consequently, ground water is
polluted to high degree by seepage from various sources
(sewage, ponds, refuse dumps, leaching of fertilizers,
pesticides from agriculture, detergent, radioactive wastes,
etc.). In our previous studies, we have investigated the
effect of disposal of untreated wastes from a pharmaceutical,
and soap and detergent industries on the emergence of
resistant bacteria in Nigeria (Lateef, 2004; Lateef and
Adewoye, 2004).
Table 1. The attributes of the commercial ice samples
a
Mean value of duplicate of 10 samples obtained from each factory within sampling period of four months.
b
Distinct bacterial isolates.
Source
T
y
pe of
ice
Uses
Microbial
load (cfu/ml)
a
Isolates
b
Factory A Bars Drinks 1.88 × 10
4
Pediococcus cerevisiae, Bacillus subtilis, Streptococcus
pyogenes, Bacillus firmus, and Pseudomonas aeruginosa.
Factory B Shaved Fish refrigeration 2.19 × 10
4
Streptococcus equi and Bacillus firmus.
Factory C Cubes Drinks 3.10 × 10
4
Streptococcus equi, Staphylococcus epidermidis,
Streptococcus pyogenes, and Micrococcus luteus.
Factory D Shaved General refrigeration 3.20 × 10
4
Micrococcus luteus and Pseudomonas aeruginosa.
40
A. LATEEF et al.

Table 2: Resistance of the bacterial isolates and their patterns
a
% Resistance obtained from the antibiogram.
b
Resistance pattern constructed from the antibiogram; antibiotic codes as defined under materials and methods.
The mean counts of heterotrophic bacteria (microbial load)
showed high presence of bacteria in all the ice samples in the
range of 1.88-3.20 × 10
4
cfu/ml. However, none of the ice
samples showed the presence of coliform as MPN of 0 was
obtained for all of them. The microbial loads fall within the
range reported by (Falcão et al., 2002) for some Brazilian ice,
but fall short of the quality reported for packaged ice obtained
in Florida, USA (Schimdt and Rodrick, 1999). The microbial
quality is also lower than the ice samples analyzed in Hong
Kong whereby only 6 out of 12 samples obtained from ice
manufacturing plants had aerobic plate count (APC) of <10
cfu/ml, while only 3 out of 89 ice samples obtained from retail
outlets had aerobic plate count of ≥1000 cfu/ml (FEHD, 2005).
The permissible levels of aerobic plate counts of ice from
manufacturing plants and retail outlets are <500 and <1000
cfu/ml respectively (Wilson et al., 1997, Nichols et al., 2000).
The presence of high APC counts in ice is an indication
of unsanitary conditions or poor hygiene practices during or
after production. In many previous studies on the quality of ice,
microbiological criteria for drinking water, similar to those
recommended by World Health Organization (WHO, 2004),
were usually applied. This is because many countries do not
have specific national microbiological guidelines for ice.
However in the United States, the International Packaged Ice
Association (IPIA) produced guidelines (IPIA, 1989) for the
industry aiming at assuring the microbiological quality of
packaged ice. These guidelines require that ice must not
contain coliforms and APC should be <500 cfu per ml. These
guidelines have been criticized to be unrealistic for all types of
ice particularly the loose ice that have undergone handling
process (Wilson et al., 1997, Nichols et al., 2000). In this
regard, it has been suggested that the APC of acceptable
loose ice should be <1000 cfu/ml. It is evident from this
study that all the ice samples analyzed, despite being
obtained from factories did not meet any of the
microbiological criteria for ice. This may not be unrelated
with the source of water used in the production of ice,
aside the unsanitary hygiene practices. We have earlier
reported high microbial load (8.0 × 10
2
-2.5 × 10
5
) of
bacteria from well water samples (Fawole et al., 2002;
Lateef et al., 2005) in Ogbomoso metropolis. Evidence
from this study suggests that the microbiological quality
of the ice samples is a cause for concern. The bacterial
isolates obtained from the ice samples include
Pediococcus cerevisiae, Bacillus subtilis, Streptococcus
pyogenes, Bacillus firmus, Pseudomonas aeruginosa,
Streptococcus equi, Staphylococcus epidermidis, and
Micrococcus luteus. It is known that most of these
organisms are pathogenic in man and capable of causing
wide range of diseases.
The resistances of the bacterial isolates to different
antibiotics and their patterns are as shown in Table 2. The
degree of resistance shown by the isolates differs ranging
from 50-87.5%, with multiple-drug resistance to 4-7
antibiotics. The cumulative resistance of all the bacterial
isolates to each antibiotic showed that all of them were
resistant to Cotrimoxazole, Ampicillin, Cefotaxine and
Cephalexin, while none was resistant to Gentamicin. The
resistance to other antibiotics ranged from 26.67% for
Ofloxacin, 66.67% for Erythromycin to 86.67% for
Bacterial isolate
Source (Ice samples from
different factories)
% resistance to the
antibiotics
a
Resistance pattern
b
M. luteus C 87.5 Tet Cot Ery Amp Cro Ofx Crl
M. luteus D 75.0 Tet Cot Ery Amp Cro Cfl
P. cerevisiae A 50.0 Cot Amp Cro Crl
B. subtilis A 75.0 Tet Cot Amp Cro Ofl Crl
B. subtilis A 75.0 Tet Cot Ery Amp Cro Crl
S. pyogenes A 87.5 Tet Cot Ery Amp Cro Ofl Crl
S. pyogenes A 75.0 Tet Cot Ery Amp Cro Crl
S. equi B 75.0 Tet Cot Ery Amp Cro Crl
S. equi C 62.5 Tet Cot Amp Cro Cfl
S. pyogenes C 62.5 Tet Cot Amp Cro Cfl
S. epidermidis C 87.5 Tet Cot Ery Amp Cro Ofl Crl
B. firmus A 62.5 Tet Cot Amp Cro Cfl
B. firmus B 75.0 Tet Cot Ery Amp Cro Cfl
P. aeruginosa A 75.0 Tet Cot Nal Amp Cro Crl
P. aeruginosa D 62.5 Cot Nal Amp Cro Crl
41
MICROBIAL QUALITY OF ICE

Tetracycline. The multiple-drug resistance obtained in this
study falls within the range that we have reported for bacterial
isolates obtained from diverse clinical, food, water, effluents
and fish samples in Nigeria (Lateef, 2004; Adewoye and
Lateef, 2004; Lateef et al., 2004; Lateef et al., 2005). The
emergence of bacteria resistant to most of the commonly used
antibiotics is of considerable medical significance (Khan and
Malik, 2001) because of the public health implications, and
there are several reports on the incidence of bacterial
resistance among bacterial isolates obtained from food
materials (Grewal and Tiwari, 1990; Singh et al., 1995; Desai
and Kamat, 1998; Khan and Malik, 2001).
As far as we know, this is the first report of work to
evaluate the antibiotic sensitivity of bacterial isolates from
edible ice, with the view of determining their public health
implications. Although consumption of ice may not in itself
represent immediate threat to personal or public health since it
is not consumed in large quantities like packaged or bottled
water, the potential for transmission of disease exists in ice
industry that is not regulated. The present study reveals that
ice may represent novel route of spread of antibiotic–resistant
bacteria especially in developing countries. As a manufactured
food, production of ice is covered by the regulations of Good
Manufacturing Practices (GMP) which address the facilities
where it is manufactured, quality of source of water and
sanitary practices during ice production. There should also be
performance of analytical tests and establishment of HACCP
to ensure microbiological safety of ice.
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