Food Protection Trends - November/December 2017 - 393

collapsible bottles to multi-purpose containers that can
handle either hot or cold beverages. Some bottles come
with built-in carbon filters that are replaceable, but not
cleanable, while others include straws and areas that are
difficult to clean properly. Some bottles have wide mouths
that make it easy to clean the interior, while others have
an opening that is only an inch or so in diameter, making
it challenging to clean the inside. Users may think that it is
sufficient to simply put the bottle into the dishwasher for
cleaning, but not all reusable water bottles are dishwasher
safe, and/or the diameter of the bottle mouth may not
permit water and detergent to enter with sufficient force to
coat the interior surface.
Improperly cleaned water bottles may present a
potential contamination risk and thus be considered a
risk for foodborne illness, particularly to those at higher
risk such as immune compromised people, older adults,
and young children. Microorganisms will normally grow
in water and on surfaces in contact with water as biofilms
(3). The availability of nutrients and lack of residual
disinfectant are some of the principal determinants of
microbial growth in drinking water (3). According to
the FDA Food Code (16), water is considered a food.
Reusable bottles are therefore food-contact surfaces
requiring proper cleaning and sanitizing. Unfortunately,
consumers may not be aware of the potential hazards
related to water bottles; thus, there is a possibility for
complacency with regard to cleaning behaviors.
It is recognized that water can be a source of disease
outbreaks (27, 42). Despite worldwide efforts and the
modern technology employed for production of safe
drinking water, transmission of waterborne diseases is still a
matter of major concern (9, 37). Some common foodborne
organisms associated with water include Campylobacter, E.
coli O157:H7, Salmonella, and Vibrio cholerae, to name a few
(7). These can lead to severe illnesses and death. Clearly,
there are health implications associated with unclean water
consumption.
Combined, the clues point to the possibility of a large
and growing problem. Reusable water bottles are growing in
popularity, but consumers may not perceive the importance
of cleaning water bottles as other food-contact surfaces
must be cleaned, which can result in careless behaviors with
regard to their cleaning. The difficulties associated with
cleaning the bottles adequately, as well as the variability
in designs and materials, make it easy to see the potential
food safety hazard. Previous studies have conducted
microbiological evaluation of bottled water (18, 22, 33, 38),
as well as cleaning protocols of infant feeding bottles (24,
26). Oliphant, Ryan, & Chu (30) studied water quality in
the personal water bottles of 75 elementary students and
stated that the use of personal water bottles for students
in elementary classrooms is not recommended because
of the significant microbial contamination levels of the

water in the bottles. However, limited information has
been published on the cleanliness of reusable water bottles
and consumer behaviors related to reusable water bottles.
Therefore, the purpose of this study was two-fold: first, to
measure contamination levels of water bottles that are in
use, and second, to understand how contamination levels
are affected by bottle usage and cleaning behaviors, by
collecting survey data from the bottle owners.
Three methods were used to assess water bottle
contamination. First was the use of adenosine triphosphate
(ATP) bioluminescence on the exterior surface of water
bottles. ATP tests provide evidence on the level of general
cleanliness (19) by measuring organic materials (41), with
results reported in terms of RLUs (relative light units).
Results are obtained rapidly but are considered only a
generalized assessment that cannot provide information
on the identity of organisms present in a sample (25).
Because of this, the second method quantified microbial
contamination through a heterotrophic plate count (HPC)
from the bottles' interior. "Heterotrophic bacteria" include
all bacteria that consume organic nutrients for growth.
These bacteria are universally present in all types of water,
food, soil, vegetation, and air (2). The heterotrophic plate
count is a means of assessing the concentration of these
bacteria in foods and water (13). Enumeration of total
heterotrophic counts is commonly used as an indicator of
overall microbiological quality (12, 32, 40), and results are
reported as colony forming units per milliliter (CFU/mL).
Finally, coliform testing was used to assess more potentially
risky coliform bacterial contamination. Coliform organisms
have long been recognized as a suitable microbial indicator
of drinking-water quality, largely because they are easy to
detect and enumerate in water (9, 40).
MATERIALS AND METHODS
This study was piloted initially to ensure adequacy of the
design, and a mixed methods approach was adopted in the
full study. The bottle exteriors were assessed for organic
contamination by ATP Bioluminescence testing. The bottle
interiors were assessed for microbial contamination by use
of Heterotrophic Plate Counts (HPC) and Coliform Plate
Counts. Lastly, the bottle owners were asked to complete
a digital questionnaire intended to document routine
behaviors related to the use and cleaning of the reusable
water bottles. Routine behaviors, bottle cleaning, and
design of the bottles (documented during the surveys) were
paired with the cleanliness assessment results through the
use of anonymous coding numbers.
A total of 90 water bottles were collected from the
participants. As shown in Table 2, 65 (72.22%) were hard
plastic bottles, 22 of which had a straw or nozzle. The third
commonly used type of water bottle was a squeezable
bottle (13.33%), followed by metal bottle (11.11%), and
glass bottle (3.33%). Built-in carbon filters were also

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