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Isolation and Characterization of Bacteria from Drinking Water Fountains at a

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DDC Professional Journal
Vol.1 No.1, September 2018
ISSN 1908-3130

Isolation and Characterization of Bacteria from Drinking Water Fountains

at a School Canteen in Davao City
Rezeile M. Degayo1, Giovanne G. Tampos1, Allan E. Bano1, Elaine Frances R. Corpuz1
Novela D. Francisco1, Maria Carmeli F. Montecillo1
Agapito M. Gabato1 and Phoebe Calica2
1
Ateneo de Davao University
2
Davao Doctors College

Abstract

Microbial testing in school facilities which students’ commonly share is critical to address
and prevent possible health issues that could arise from failing to understand the environment of
different microorganisms. This research tested microbial organisms in drinking water fountains and
hand wash faucets at a school canteen in Davao City. Morphological and biochemical tests were
performed to characterize the isolates. Pseudomonas aeruginosa and Coagulase Negative
Staphylococcus (CoNS) were isolated from water fountains where most if not all students are using.
The focus of the study was to identify any possible bacteria present in one of the water fountain in
the school campus namely, in the school’s canteen. The researchers in the study recommends that
the future researchers should also consider other water fountain present inside the campus for
further investigation on the presence of bacteria other than what has been discovered. Moreover,
biochemical methods have been used to identify the bacteria isolates in the study: Pseudomonas
aeruginosa which is a frequent cause of pneumonia, urinary tract infections and bacteremia, and
Coagulase Negative Staphylococcus which can cause endocarditis and urinary tract infections. The
researchers in this study suggest that to further validate the identified isolates, the use of Molecular
Tests like PCR is highly recommended. Furthermore, given the results are identified infectious
bacteria in the school’s water fountain, the management should therefore immediately address this
concern to prevent possible health issues that may arise. Water treatment and proper sanitation on
fountains should be implemented to help alleviate the problem concerning the presence of bacteria.

Keywords: Pseudomonas aeruginosa, Coagulase Negative Staphylococcus (CoNS), water

fountains, biochemical tests, morphological characterization

Corresponding email: giovtampos@gmail.com

Introduction

Microbial communities inhabit in and even in drinking water fountains

some unexpected environments such as in
school facilities which are commonly shared
by students: classrooms, canteens and sources
of drinking water. Bacterial communities are
found inhabiting classroom surfaces(Meadow
et al., 2014), food in canteens (Pandey, 2016)
(Burkowska-But, Swiontek Brzezinska, &
Walczak, 2013). Frequent and direct contact
to these microbes may result to different
severity of sickness like acute gastroenteritis
(Altzibar et al., 2015), giardiasis and

Rezeile M. Degayo et al. 2018 14

DDC Professional Journal
Vol.1 No.1, September 2018
ISSN 1908-3130
cryptosporidiosis (Eisenstein, Bodager, &
Ginzl, 2008).
To some extent, human aids in the
dispersal of different microbial organism in
indoor surfaces like classrooms. Transfer of
bacterial assemblages is possible through
human skin and surface contact. However,
most bacteria in human body are not
pathogens. They only exists as commensal
component of our own microbiome (Meadow
et al., 2014).
Consumption of contaminated food
with microorganisms causes several food
borne diseases. Food borne illness are
primarily caused by microbial pathogens,
microbial toxic products, and poisonous
chemicalsin contaminated restaurants and
canteens (Pandey, 2016). Improper handling
and preparation of food and without proper
sanitation guidelines brought serious hazard
to human health due to possible food
contamination.
Schools’ water fountains could also
host several microbial communities. Fecal
coliform is one of the sources that
contaminate drinking water. Recent studies
also revealed that there are some bacterial
organism which contain plasmid from
different microorganism and have developed
resistance to some antibiotics (Gomes Freitas
et al., 2017). Antibiotic-resistant
microorganisms are now becoming a serious
public concern.
Thus, microbial testing in school
facilities which students’ commonly shared is
critical to address and prevent possible health
issues that could arise from failing to
understand the environment of different
microorganisms. This research tested
microbial organisms in drinking water
fountains and hand wash faucets as a school
canteen in Davao City.
Rezeile M. Degayo et al. 2018
Materials and Methods
Media Preparation
Five culture media were prepared to
cultivate and isolate the microorganisms
within the target sites. These media include
Maconkey Agar, Blood Agar Plate (BAP),
Chocolate Agar, Nutrient Broth, and
Sabouraud Dextrose Agar (SDA). Triple
sugar iron agar (TSI), Lysine Iron Agar
(LIA), Citrate, and Sulfide, Indole, and
Motility (SIM) were used on further work up
and identification of the bacterial
microorganisms isolated.
Study Sites
Due to time and material constraints,
samples were only collected to five water
sources within the selected school campus in
Davao City. However, the sites were only
limited to canteen areas which include two
drinking fountains and three faucets at wash
area. These sites were collected since almost
all students including faculty share this area.
Isolation and Cultivation of Bacteria
The purpose of bacterial cultivation is
to grow and isolate all bacteria present in our
chosen sample site. And to determine which
of the bacteria that grow are most likely
causing infection and which are likely
contaminants, and to obtain sufficient growth
of clinically relevant bacteria to allow
identification and characterization.
Preparation of Bacterial Smear and Gram
Staining
Differential stains are very useful in
microbiology because they allow the
classification of bacteria. For example, the
gram stain classifies bacteria as either Gram-
Positive or Gram- Negative. Basically, the
differential stains consist of the following
technique with some amount of variation.
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DDC Professional Journal
Vol.1 No.1, September 2018
ISSN 1908-3130

Staphyloccus (H2O2). It is used to differentiate those

After studying cultures on agar plates, bacteria that produce an enzyme catalase,
and colonies was gram stained, Gram Positive such as staphylococci, from non-
cocci was noted. Catalase test and Coagulase catalase producing bacteria such as
test followed to differentiate coagulase- streptococci.
positive from Coagulase Negative
staphylococcus. Staphylococci are among the Coagulase Test (Tube)
ubiquitous organisms. They are part of the This version of the coagulase test is
normal flora of man. Staphylococci are used to identify the presence of either bound
responsible for over 80% of the suppurative coagulase or free coagulase, which is an
diseases encountered in medical practice.The extracellular enzyme. The coagulase test is
genus staphylococci have three (3) commonly useful for differentiating potentially
recognized species: S. aureus, S. epidermidis, pathogenic Staphylococci such as
and S. saphrophyticus. Among these, S. Staphylococcus aureus from other Gram
aureus is the most significant pathogen to positive, catalase-positive cocci.
man, although S. epidermidis and S.
saphrophyticus are also associated with Biochemical Test
human infections. Biochemical tests are the tests used
for the identification of bacteria species based
Catalase test on the differences in the biochemical
This test demonstrates the presence activities of different
of catalase, an enzyme that catalyses the bacteria. Bacterial physiology differs from
release of oxygen from hydrogen peroxide one species to the other.

Figure 1.Catalase, coagulase and other biochemical tests for bacteria.

Rezeile M. Degayo et al. 2018 16

DDC Professional Journal
Vol.1 No.1, September 2018
ISSN 1908-3130

Results positive. Further at SIM, it was found non-

After 24 hours and 48 hours of
incubation (Table 1 and 2), culture media
plates were examined and described. The
appearance, shape, and texture of the colonies
observed were recorded. Growth from BAP
was gram stained and examined on the
microscope under oil immersion field.
Sample 1, 2 and 4 showed significant growth.
Based on the gram stain results, Sample 1
showed gram positive rod-shaped bacilli
while samples 2 and 4 showed gram positive
cocci. Gram negative bacilli were further
inoculated to triple sugar iron, lysine iron
agar, citrate, and sulfide, indole, and motility
tube.
Biochemical test on tubes are mainly
used to identify gram negative bacilli species
(Table 3). The ability of the bacteria to react
on those biochemical media is one of the
bases for the identification. On TSI, the result
showed alkaline on the butt and alkaline on
the slant with no presence of sulfide and gas.
Sheen was also noted. LIA showed no change
in color on the other hand and was interpreted
as deaminase negative decarboxylase
positive. On citrate, from original color green
it turned into blue which was noted as
motile with no sulfide present and was indole
negative. Based on the biochemical results, it
was suggestive to be a Pseudomonas
aeruginosa species. Mueller Hinton Agar
confirmed the result species when it
developed a green pigment on the media.
Sample 2 and 4 was tested on catalase
and coagulase since gram staining result
showed a gram positive cocci organism.
Catalase test is used to differentiate
Staphylococcus species from Streptococcus
species. Catalase test showed positive result
for the colonies from sample 2 and 4.
Production of bubbles is an indicative of a
positive reaction. Coagulase test was then
performed since we already know that it was
a Staphylococcus species. Coagulase test will
show if the organism is a S. aureusor a
Coagulase Negative Staphylococcus (CNS)
species. After 24 hours of incubation on
Coagulase test, both sample 2 and 4 failed to
develop fibrin clot. It suggested that the result
was negative. Unavailability of materials to
further identify the microorganism is a major
constraint hence the species was only
identified as a Coagulase Negative
Staphylococcus (CNS).

Table 1. Observations to culture media after 24 hours of incubation.

Specimen Maconkey Blood agar Chocolate Sabouraud Nutrient Gram

Agar Plate Agar Dextrose Broth Stain
Agar
Sample 1 Very Light Very Light Very Light No Growth Cloudy Gram-
Growth of Growth of Growth of negative
Non Dirty White, Dirty White Rods
Lactose and translucent and translucent
Fermenter colonies colonies

Sample 2 No Growth Very Light Very Light No Growth Clear

Growth of Growth of
White and White and
round colonies round colonies

Rezeile M. Degayo et al. 2018 17

DDC Professional Journal
Vol.1 No.1, September 2018
ISSN 1908-3130

Specimen Maconkey Blood agar Chocolate Sabouraud Nutrient Gram

Agar Plate Agar Dextrose Broth Stain
Agar
Sample 3 No Growth Presence of Presence of No Growth Clear
Contaminants Contaminants

Sample 4 No Growth Very Light Very Light No Growth Cloudy Gram

Growth of Growth of Positive
White, Round White, round, Cocci
and milky and milky
colony colonies
Sample 5 No Growth No Growth No Growth No Growth Clear

Table 2.Observations on culture media after 48 hours of incubation.

Specimen Maconkey Blood agar Chocolate Sabouraud Nutrient Gram

Agar Plate Agar Dextrose Broth Stain
Agar
Sample 1 Re-isolate: Light Growth Light Growth No Growth Cloudy Gram-
Moderate of Dirty of Dirty negative
Growth of White, and White and Rods
Non Lactose translucent translucent
Fermenter colonies colonies

Sample 2 No Growth Light Growth Light Growth No Growth Slightly Gram

of White and of White and Cloudy Positive
Round round Cocci
colonies colonies

Sample 3 No Growth Presence of Presence of No Growth Clear

Contaminants Contaminants

Sample 4 No Growth Light Growth Light Growth No Growth Slightly Gram

of White, of White, Cloudy Positive
Round and round, and Cocci
milky colony milky
colonies
Sample 5 No Growth No Growth No Growth No Growth Clear

Rezeile M. Degayo et al. 2018 18

DDC Professional Journal
Vol.1 No.1, September 2018
ISSN 1908-3130
Table 3. Biochemical test results on Gram Negative Rod-shaped bacteria
TSI LIA
Slant Butt Gas H2s Slant Slant
Lysine Lysine
Deam Decarb
K K NEG NEG NEG POSITIVE
Note:
w/
sheen
Legend: K=Alkaline
Discussions
P. aeruginosa is an aerobic gram-
negative bacterium and a typified by motile,
non-spore forming rods that are oxidase
positive and lactose nonfermenters. It is a
member of the genus Pseudomonas,
colloquially called the pseudomonads. The
water-soluble pigments, pyocyanin and
pyoverdin, are responsible for the distinctive
blue-green color on solid media. The presence
of polar flagella and pili gives P. aeruginosa
motility (Fujitani et al., 2011).
It is widespread in the environment,
particularly in a variety of water sources such
as hospital (Lefebvre et al., 2017) and
municipal drinking water systems (Felföldiet
al., 2010), healthcare facilities (Lefebvre et
al., 2017; Anaissie et al., 2002),
accommodation facilities (Huhulescuet al.,
2011), as well as in swimming pools and hot
tubs (Guidaet al., 2016; Amaglianiet al.,
2012]. It is also a major cause of skin
infections such as folliculitis and external
otitis (Berthelot et al., 2005; Mena et al.,
2009). In water systems, P. aeruginosa has
the ability to grow (Blanc et al., 2004;
Hunter, 1993) and form biofilms
(Rasamiravaka et al., 2015).
Ray et al. (2016) conducted a study to
find out the extent of germs present in hand of
school children. Although they washed hands
Rezeile M. Degayo et al. 2018
CITRATE SIM
Slant and Sulfide Indole Motility
Butt
POSITIVE NEGATIVE NEGATIVE NON
MOTILE
before meals, they hardly used soap due to
non-availability of soap and contaminated
water and surfaces in the food industry
becomes potential source of P. aeruginosa
infections. Study demonstrated the presence
of pathogenic microorganisms on the hands
of 61% of the children including P.
aeruginosa (3%).
Schiavano et al. (2017) analyzed the
prevalence, antibiotic resistance and genetic
relatedness of P. aeruginosa isolates obtained
from potable and recreational water samples.
Their study has revealed the presence
of P. aeruginosa in different water samples,
including resistant strains, especially in
swimming pools.
Costa et al. (2015) analysed
Nosocomial outbreak of P. aeruginosa
associated with a drinking water fountain.
This study revealed a drinking water fountain
contaminated withP. aeruginosa. The
drinking water fountain was used for the
alimentation by percutaneous endoscopic
gastrostomy and became the origin of the
outbreak.
P. aeruginosa is a pathogen frequently
implicated in healthcare-associated infections
(HAIs), particularly in critically ill or
immunocompromised patients (Bodey and
Jadeja, 1985; Chatzinikolaouet al., 2000). It is
19
DDC Professional Journal
Vol.1 No.1, September 2018
ISSN 1908-3130
a versatile pathogen with the ability to cause
diverse infection types. Data from the
National Nosocomial Infections Surveillance
system from 1986–2003 reported P.
aeruginosa as the second most common cause
of pneumonia (18.1%), the third most
common cause of urinary tract infection
(16.3%) and the eighth most frequently
isolated pathogen from the bloodstream
(3.4%) (Gaynes et al., 2005).
Coagulase Negative Staphylococcus
(CoNS) are gram-positive cocci that divide in
"grape-like" clusters and are catalase-positive.
CoNS are readily recovered from biologic
specimens with use of commercially-
available automated blood culture systems or
standard solid or broth media. Their main
ecological niches are skin and mucous
membranes of humans and animals, and they
are therefore always in a very close, and
mainly symbiotic, relationship with their
natural hosts.CoNSare found as food-
associated saprophytes also while many other
CoNS species colonize the skin and mucous
membranes of humans and animals and are
less frequently involved in clinically
manifested infections.
Most of the CoNS species are S.
epidermidis which may cause urinary tract
infection, bacterial endocarditis, septicemia
and wound infection and S.
saprophyticuswhich cause a community-
acquired urinary tract infection.
Conclusion
Two microbial organisms,
Pseudomonas aeruginosa and Coagulase
Negative Staphylococcus (CoNS) were
isolated from water sources in a school
canteen in Davao City. The alarming result
was the location where these microbial
organisms were isolated. These
microorganisms were found from the two
water fountains where most if not all students
are using.
Rezeile M. Degayo et al. 2018
Recommendation
The focus of the study was to identify
any possible bacteria present in one of the
water fountains in the school campus. The
researchers in the study recommended that
the future researchers should also consider
other water fountains present inside the
campus for further investigation on the
presence of bacteria other than what has been
discovered. Moreover, biochemical methods
have been used to identify the bacteria
isolates in the study: Pseudomonas
aeruginosa and Coagulase negative
staphylococcus. The researchers in this study
suggested that to further confirm the identity
of the isolates, the use of molecular tools
(PCR) is highly recommended.
Furthermore, given the results are
infectious bacteria found in the school
campus, the management should therefore
immediately address this concern to prevent
possible health issues. Water treatment and
proper sanitation on fountains should be
implemented to help alleviate the problem
concerning the presence of bacteria.
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