TOPS OF BEVERAGE CANS ARE A POTENTIAL SOURCE OF INFECTION: A STUDY OF BACTERIAL LOAD PRESENT ON THE LIDS OF BEVERAGE CANS

Abstract

It is common practice to drink directly from tops of beverage cans which are exposed to environmental contaminants during handling and storage. The purpose of this research was to determine the bacterial load present on the lids of beverage cans and find out the ways to significantly reduce the number of bacteria present on them. One hundred and eighty apparently clean and non-refrigerated beverage cans were collected from different shops and divided into two groups. First group was used for cleaning experiments and second group was used to determine the effect of refrigeration on bacterial load. Different types of bacteria were isolated which belonged to Bacillus spp, Staphylococcus spp, Corynebacterium spp, Streptococcus spp, Klebsiella spp and Escherichia spp. AST result pattern of S. aureus isolates did not show the presence of MRSA (Methicillin-resistant S. aureus) and MSSA (Methicillin-susceptible S. aureus). E. coli isolates were found to be highly sensitive to ceftriaxone and highly resistant to erythromycin. Cleaning with tap water plus wiping with dry tissue resulted in maximum removal of bacterial load (76.6%) as compared to other methods (68.8% with dry tissue and 47.3% with tap water) and is thus the most effective method for this purpose. Refrigeration lowered the bacterial load by 16.6%, but it depends upon the type of bacteria present. Beverage cans are present in different environments and are handled by different people and thus can be a potential source of infection for the

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The Journal of Microbiology and Molecular Genetics (JMMG)
Vol. 3, No. 2 (2022), pp. 49-61
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TOPS OF BEVERAGE CANS ARE A POTENTIAL SOURCE OF
INFECTION: A STUDY OF BACTERIAL LOAD PRESENT ON THE LIDS
OF BEVERAGE CANS
Saleha Hafeez1 and Shahida Hasnain2*
1Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences, National University of
Sciences and Technology, Islamabad, Pakistan
2Institute of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan.
*Corresponding author Email: shafeez.phdabs19asab@student.nust.edu.pk
Abstract
It is common practice to drink directly from tops of beverage cans which are
exposed to environmental contaminants during handling and storage. The purpose
of this research was to determine the bacterial load present on the lids of beverage
cans and find out the ways to significantly reduce the number of bacteria present
on them. One hundred and eighty apparently clean and non-refrigerated beverage
cans were collected from different shops and divided into two groups. First group
was used for cleaning experiments and second group was used to determine the
effect of refrigeration on bacterial load. Different types of bacteria were isolated
which belonged to Bacillus spp, Staphylococcus spp, Corynebacterium spp,
Streptococcus spp, Klebsiella spp and Escherichia spp. AST result pattern of S.
aureus isolates did not show the presence of MRSA (Methicillin-resistant S.
aureus) and MSSA (Methicillin-susceptible S. aureus). E. coli isolates were found
to be highly sensitive to ceftriaxone and highly resistant to erythromycin. Cleaning
with tap water plus wiping with dry tissue resulted in maximum removal of
bacterial load (76.6%) as compared to other methods (68.8% with dry tissue and
47.3% with tap water) and is thus the most effective method for this purpose.
Refrigeration lowered the bacterial load by 16.6%, but it depends upon the type of
bacteria present. Beverage cans are present in different environments and are
handled by different people and thus can be a potential source of infection for the

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consumers. It is highly recommended that beverage cans should be cleaned before
drinking.
Keywords
Beverage cans, Bacterial load, Consumer, Antibiotics, Refrigeration
INTRODUCTION
Usage of beverage cans is increasing day by day and people are in a habit of drinking directly
from the cans. This can lead to serious consequences as the lid is highly contaminated with
microorganisms (Fekete, 2018; Gündüz et al., 2019). The contamination of beverage cans before
arriving to the customer can occur anywhere from the point of manufacturing to the point of
storage in the stores. These cans are stored in the refrigerator or displayed on the shelves with
lids uncovered. Thus, exposing the lids to the dirt and germs that will directly come into contact
with the consumer’s mouth (Abraham et al., 2018; Dawson et al., 2018).
It is to be noted that the dirt is not the only culprit of contamination here. Beverage cans before
arriving to the customer can also be contaminated from unhygienic handling, insects and rodents
(Gündüz et al., 2019). Places which have drainage areas often contain moisture which attracts
many insects and rodent species. Among insects, cockroaches are the most important reservoirs
of infectious pathogens such as Salmonella spp that can contaminate food products. Dust can
also harbor Salmonella spp which can remain viable there for up to 10 months (Michaels et al.,
2003). Rats are notorious for about 35 rat-borne diseases of which some are directly transmitted
from the feces, urine, saliva and bites from rats while some are indirectly transmitted through an
arthropod vector. Important rat-borne disease includes bacterial zoonotic diseases such as
bubonic plague commonly known as Black Plague, Leptospirosis, Salmonellosis and many viral
zoonotic diseases (CDC, 2010).
No case of illness caused by drinking directly from the dirty beverage cans have been reported in
Pakistan. This could be due the fact that people usually consume refrigerated beverage cans
which may reduce the contamination level and also most people use straws or prefer to use glass
or cup to drink beverages which prevents the direct contact with the lids of beverage cans.
The aim of this study was to determine the hygienic conditions of the lids of beverage cans,
compare different cleaning methods such as wiping with dry tissue, rinsing under running tap

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water and rinsing with tap water plus wiping with dry tissue, determine the antibiotic
susceptibility pattern of isolated S. aureus and E. coli isolates against commercially available
antibiotics.
MATERIAL AND METHODS
Sample collection
A total of 180 apparently cleaned, non-refrigerated beverage cans were collected (convenience
sampling) in sterilized plastic zipper bags to avoid environmental contamination from different
retail shops, departmental stores and drink corners. These cans were divided into 2 groups. First
group contained 150 cans which were used for cleaning experiment and second group contained
30 cans which were used to check the effect of low temperature on bacterial load.
Detection of bacterial diversity and antibiotic susceptibility testing
Types of bacterial were identified in all 180 cans. Bacterial groups were identified through
biochemical testing and by the scheme provided in Bergey’s Manual of Determinative
Bacteriology. Antibiotic susceptibility test AST was performed on S. aureus and E. coli isolates
by Kirby-Bauer disc diffusion method on Muller Hinton Agar MHA.
Cleaning experiment
For cleaning experiment cans of first group were subdivided into 3 groups in such a way that
each subgroup contained 50 cans (labelled as dry tissue paper, tap water and tap water plus dry
tissue paper). Each group was cleaned with sterilized dry tissue paper, rinsed with tap water (30
seconds) and rinsed with tap water (30 seconds) plus cleaned with dry tissue paper. The lid of
each can including the mouthpiece (area where mouth makes contact with can) was divided into
two halves. One half was swabbed before cleaning and whole lid was swabbed after cleaning
(Figure 1a-b). It was assumed that bacteria were uniformly distributed over each lid.
Effect of refrigeration
To check the effect of refrigeration on bacterial load above-mentioned protocol was followed on
second group cans with slight modifications (one half was swabbed before refrigeration and
whole lid was swabbed after refrigeration at 4°C for 24 hours) as shown in Figure 1a and 1b.

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Figure 1a: Grid area was used to take sample from the beverage cans before cleaning and refrigeration.
Figure 1b: Grid area was used to take sample from the beverage cans after cleaning and refrigeration.
Statistical analysis
Paired t-test was applied to detect the difference before and after cleaning. P-value ≤ 0.05 was
taken as significant.

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RESULTS
Detection of bacterial diversity
In this study apparently clean and non-refrigerated beverage cans were used. Of the total 180
beverage cans, Gram positive bacteria were found to be present on all samples (100%) while
Gram negative bacteria were found only on 48 (26.6%) samples. Among identified Gram
positive bacteria, Bacillus spp were distributed on all samples (100%), followed by
Staphylococcus spp on 168 (93.3%) samples, Corynebacterium spp on 11 (6.1%) and
Streptococcus spp on 4 (2.2%) samples. Among Staphylococcus spp, S. aureus were present on
all 168 (100%) samples. Among Gram negative bacteria, Escherichia spp mainly E. coli were
found on all 48 (100%) samples, followed by Klebsiella spp 13 (27%) and no Salmonella spp
and Pseudomonas spp were detected.
Antibiotic susceptibility testing
The antibiotic susceptibility pattern of all E. coli isolates (N = 48) showed highest sensitivity to
ceftriaxone 48 (100%), amikacin 40 (83.3%), ciprofloxacin 39 (81.2%) ampicillin 37 (77.1%),
the lowest sensitivity was observed in erythromycin 19 (39.6%), trimethoprim/sulfamethoxazole
35 (72.9%) and chloramphenicol 36 (75%). The antibiotic susceptibility pattern of all S. aureus
isolates (N = 168) showed highest sensitivity to vancomycin 168 (100%), linezolid 168 (100%)
and trimethoprim/sulfamethoxazole 145 (86.3%), the lowest sensitivity was observed in
erythromycin 56 (33.3%), and ciprofloxacin 60 (35.7%). Antibiotic susceptibility patterns of S.
aureus and E. coli isolates are summarized in Table 1a and 1b, respectively.
Table 1a: Antibiotic susceptibility pattern of S. aureus isolates isolated from lids of beverage
cans.
Antibiotic Class
Antibiotics
Susceptibility Pattern
Sensitive
N (%)
Intermediate
N (%)
Resistant
N (%)
Antifolate/
Sulfonamides
Trimethoprim/
Sulfamethoxazole
145 (86.3%)
14 (8.3%)
9 (5.4%)
Cephalosporin
Cefoxitin
123 (73.2%)
45 (26.7%)
0
Glycopeptide
Vancomycin
168 (100%)
0
0

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Lincomycin
Clindamycin
116 (69%)
30 (17.9%)
22 (13.1%)
Macrolide
Erythromycin
56 (33.3%)
0
112 (66.7%)
Penicillin
Ampicillin
60 (35.7%)
0
108 (64.3%)
Oxazolidinones
Linezolid
168 (100%)
0
0
Table 1b: Antibiotic susceptibility pattern of E. coli isolates isolated from lids of beverage cans.
Antibiotic Class
Antibiotics
Susceptibility Pattern
Sensitive
N (%)
Intermediate
N (%)
Resistant
N (%)
Aminoglycosides
Amikacin
40 (83.3%)
8 (16.7%)
0
Antifolate/
Sulfonamides
Trimethoprim/
Sulfamethoxazole
35 (72.9%)
0
13 (27.1%)
Cephalosporin
Ceftriaxone
48 (100%)
0
0
Fluoroquinolones
Ciprofloxacin
39 (81.2%)
9 (18.8%)
0
Macrolide
Erythromycin
19 (39.6%)
0
29 (60.4%)
Penicillin
Ampicillin
37 (77.1%)
0
11 (22.9%)
Phenicol
Chloramphenicol
36 (75%)
4 (8.3%)
8 (16.7%)
Cleaning experiment
Bacterial load was significantly reduced by 68.8% (P = 0.0002), 47.3% (P = 0.0005) and 76.7%
(P = 0.0001) after cleaning with dry tissue paper, rinsing with tap water and rinsing with tap
water plus cleaning with dry tissue paper respectively as shown in Table 2 and Figure 2.
Table 2: Comparison of different cleaning methods and effect of refrigeration on the number of
bacterial colonies.
Group
No. of Colonies
Percentage Cleaned
P-value
Before Cleaning
After Cleaning
Dry Tissue
3154
582
68.8
0.00021

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Paper
Tap
Water
2906
968
47.3
0.00050
Tap Water + Dry
Tissue Paper
2871
378
76.7
0.00010
Before
Refrigeration
After
Refrigeration
Percentage
Reduction
Non-refrigerated
Cans
3139
2245
16.6
0.0038

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Figure 2: Comparison of bacterial load before and after treatment where A is cleaning with dry tissue, B is cleaning
with tap water, C is cleaning with tap water plus dry tissue and D is effect of refrigeration
Effect of refrigeration
Beverage cans were refrigerated to check the effect of low temperature on the number of bacteria
present on the lids. Bacterial load was reduced by 16.6% (P = 0.0024) and was observed in all
samples (100%) as shown in Table 1 and Figure 2.
DISCUSSION
In this study, apparently clean and non-refrigerated cans were collected from different shops and
from different locations. All of these cans were found to be contaminated with a variety of
bacterial species. Isolated bacteria belonged to Bacillus spp, Staphylococcus spp,
Corynebacterium spp, Streptococcus spp, Escherichia spp and Klebsiella spp. Identified bacteria
were S. aureus and E. coli. The presence of different types of contaminants indicated that the
bacterial load present on the lids of cans mainly depends on the environment in which they are

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placed and also on the microbial flora of the human handling them. Similar bacteria were
reported on lids of beverage cans (Michaels et al., 2003), other inanimate objects such as
keyboards mobile phones (Koscova, Hurnikova, & Pistl, 2018), ICU equipments (Nazeri et al.,
2019) and Automated Teller Machines ATM (Dawodu & Akanbi, 2021).
S. aureus is the normal flora of human and is present on objects which are frequently touched by
human hands (Bhatta et al., 2018), indicated the possible human handling of the beverage cans.
A study published by Domon et al. in (2015) reported that MRSA and MSSA cannot survive on
inanimate dry objects such as on shopping baskets. The AST results of present study did not
show the presence of MRSA and MSSA on the lids of beverage cans which are in agreement
with the above-mentioned study. This indicates that S. aureus are likely to be transmitted from
the healthy human handlers.
Presence of E. coli is considered the indicator of fecal contamination, suggesting the possible
contamination through the unhygienic conditions of person handling them (Abraham et al.,
2018). According to Centers for Disease Control and Prevention CDC depending upon the type
of strain, ingestion of E. coli can cause wide range of symptoms including bloody diarrhea, fever
and vomiting (CDC, 2021). Drinking directly from the beverage cans contaminated with E. coli
can cause serious infection. In present study AST pattern of E. coli against antibiotics ceftriaxone
(N = 48, 100%), amikacin (N = 40, 83.3%), ampicillin (N = 37, 77.1%), chloramphenicol (N =
36, 75%), and ciprofloxacin (N = 39, 81.2%) follow almost the same pattern reported by
Fratamico, et al. in (2008), who reported the sensitivity of shiga toxin-producing E. coli (N =
219) isolated from swine feces against antibiotics amikacin, ceftriaxone, ampicillin,
chloramphenicol, and ciprofloxacin as 100%, 99.5%, 84.9% 75.3%, and 99.5%, respectively
(Fratamico et al., 2008).
In order to reduce the bacterial load, three different cleaning methods were employed. A study
published by Michaels et al. (2003) showed the most effective method to remove contamination
from tops of food and beverage cans is rinsing with tap water and then wiping with paper towel.
Wiping with paper towel did not show any better results than rinsing with tap water and wiping
with moist tissue paper (Michaels et al., 2003). The results of present study showed significant
reduction of bacterial load in all three cleaning methods and are in agreement with the above-
mentioned study but did not follow the same trend. Cans that were first rinsed with running tap

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water and then wiped with dry tissue showed good results followed by wiping with dry tissue
and rinsing with tap water. The possible explanation in present study is tissue paper which were
already sterilized and packed in plastic wrappers and were opened only when required for
cleaning. Dry tissue papers use friction to mechanically remove the dust and contaminants from
the lids (Michaels et al., 2003), while tap water use high washing flow to mechanically detach
bacteria (Uhlig et al., 2017) thus, reduced the already present bacterial load. Another explanation
is that there could be a difference of bacterial populations in tap waters used in our study and the
mentioned above. Bacteria are also present in water (Cabral, 2010). It is difficult to detect the
exact quantity in running tap water. At one time there is no microorganism present in it but at
other times it is loaded with huge quantity and variety of microorganisms. Rinsing with water in
addition to removing must have added more and different bacteria on beverage cans but not to
the point where the bacterial load was increased from the original value.
In countries like Pakistan where the weather is hot, refrigerated beverage cans are consumed. To
check whether low temperature has any effect on the bacteria present on the lids of beverage
cans bacterial load was determined before and after refrigeration. Effect of low temperature on E.
coli introduced on the lids of beverage cans has been published by Dawson et al. (2018) in which
they reported the decrease in E. coli counts after prolonged storage at refrigeration temperature.
They also reported that E. coli counts decrease more rapidly in cans placed under room
temperature compared to cans placed at low temperature due to the retention of moisture. Similar
results have been reported by Wilks, Michels, and Keevil. (2005), who showed the survival of E.
coli O157 on metal surfaces at different temperatures and time. In present study, refrigeration
lowered the bacterial load to some extent. The possible explanation of reduction in the bacterial
load after refrigeration for 24 hours is that bacteria living in the environment are challenged by
different stressors (Batool, Yrjälä, & Hasnain, 2014). In the case of beverage cans these stressors
are low nutrient levels and low water content. Beverage cans during refrigeration faced another
stress which was low temperature or cold stress. Refrigeration favors the growth of
psychrotrophs (Tatini and Kauppi, 2002). According to the Food Safety Information provided by
United States Department of Agriculture Food Safety and Inspection Service USDA revised in
May 2010, refrigeration slows bacterial growth. But because bacteria were already present in the
stressful environment, addition of another stress (cold stress) must have killed some of the

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bacteria and hence the bacterial load was reduced. But it depends upon the type of bacteria
present.
CONCLUSION
Beverage cans are exposed to different environmental contaminations including handling by
different people and are thus loaded with different types of microorganisms which can be a
potential source of infection for the consumers. It is therefore highly recommended that lids of
beverage cans should be cleaned before directly applying mouth to them for drinking.
Acknowledgment
We are thankful to the Department of Industrial Biotechnology, Atta-ur-Rehman School of
Applied Biosciences (ASAB) National University of Sciences and Technology (NUST) for their
support and services.
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