A Comparison of Microbes Across Various
Water Fountains
Aliyah Barreiro, Caroline Kmiecik, Emily Foster, Emilly Hancock, Madysson Volkman, Michael Nelson,
and Vernon Gradney
Abstract (Emily Foster)
Many people regularly use water provided in public spaces. In many places, such as a school, the
people attending should not have to worry whether the water that they consume is clean. All
water sources, including water fountains should be kept clean. In this study, five water fountains
on the third floor of UHCL’s Bayou Building were swabbed to compare the isolates found on the
fountains. Bacteria were isolated and identified with MALDI-TOF, and antibiotic resistance
testing was performed. A cluster analysis was performed, and there were no clear clusters found
across the fountains. Penicillin resistance was found in WF06, WF07, and WF35.
Chloramphenicol resistance was found in WF07. Polymyxin B resistance was seen in WF04,
WF06, WF07, and WF20. The WF07 isolate was resistant in all three antibiotics, which indicates
multi-drug resistance. Our findings show that the bacteria are from a single source that gets
distributed via the plumbing system. In the future, routine monitoring of the water fountains and
sanitation protocols can be put in place.
Keywords: Bacterial identification, antibiotic resistance, water fountain
Introduction (Emily Hancock)
Many people who drink out of water fountains assume it is clean of harmful bacteria, but that is
not always the case. Waterborne illness is often the effect of a contaminated water source,
biofilms in the pipes, or human transmission [8]. Water fountains can harbor opportunistic
pathogenic microorganisms like Brevundimonas sp. and Escherichia coli (E.coli). E. coli is
usually harmless to humans, but can be opportunistic pathogens to those who are
immunocompromised and come in contact with an opportunistic strain. It is non-spore-forming,
motile, Gram-negative bacilli (rod-shaped) that is transmitted through fecal/oral contact [5].
When someone becomes infected with E.coli, the main symptoms are diarrhea, vomiting, and
fever [8]. Sewage contamination of an inadequately chlorinated water source can spread E.coli
to those drinking from it. A study reported by O'Mahony et al. [6] showed that 251 passengers
and 51 crew members became ill with gastroenteritis (an intestinal infection with symptoms of
watery diarrhea) after consuming tap water on a cruise ship that was supplied from a water
source contaminated with sewage and Enterotoxigenic Eschericia coli. According to the EPA,
the Maximum Contaminant Level Goal (MCLG) is zero. This is the maximum level the
contaminant can be at without causing adverse health effects. If levels exceed this amount, the
public must be notified [7].
Brevundimonas sp. are aerobic non-fermenting, Gram-negative opportunistic pathogenic bacteria
that can cause infections that are invasive and severe. Brevundimonas sp. can stick to the walls of
cast iron pipes and utilize elements the pipes release during degradation, like phosphorus, for a
source of nutrients to promote growth [3]. If the bacteria is found in pipes connected to a source
of tap water, it can slough off into the water and cause serious infection in immunocompromised
people consuming it. Brevundimonas sp. is resistant to fluoroquinolones [4] and more likely to
cause infection in people with compromised immune systems vs. healthy individuals. Different
species of the genus Brevundimonas cause different illnesses with different levels of severity. For
example, the species Brevundimonas diminuta is classified as having relatively low virulence [9]
because of the low number of reports of serious infection but they can also cause infections that
are invasive and severe [9]. A study on cancer patients at the University of Texas M.S. Anderson
Cancer Center in Houston,TX from 1998-2004 revealed seven patients were infected with B.
diminuta and it caused serious symptoms [4]. They were shown to exhibit high fevers up to
39.2℃ and infections of bloodstream, intravascular catheter, urinary tract, and pleural space [4].
The bacteria could have been present in the pipes that supply drinking water to the hospital
which led to patient exposure. The prophylactic use of quinolones in six of the seven patients
tested also made the patients more susceptible to infection by fluoroquinolone-resistant bacteria
[4]. Because of B.diminuta resistance to fluoroquinolones, the doctors of the patients in this case
study had to find alternative treatment options.
In high volume areas, like schools and hospitals, bacteria are more likely to be found. This could
be because of poor hygiene, poor disinfection techniques, or contaminated water distribution
systems. In order to monitor bacteria levels in water fountains, pipes and water sources should be
routinely tested for opportunistic, pathogenic bacteria, and water system equipment should be
replaced or disinfected accordingly. If the same bacteria is found in many different samples, a
contaminated water source is likely the cause. However, if there are large amounts of one type of
bacteria in one place, the cause is more likely to be human transmission.
In this study, samples were taken from five different water fountains to test for bacterial
contamination and compare the variability of bacteria found in each water fountain site. They
were then cultured to isolate the colonies and identified through the use of MALDI-TOF MS and
Kirby Bauer Test.
Materials and Methods (Michael Nelson and Vernon Gradney)
Sampling and Culturing
Five water fountains on the third floor of UHCL’s Bayou Building were selected to sample for
the presence of bacteria (Figure 1). The water fountains that were then selected (B35, B36 etc.)
were swabbed by new sterile swabs that were directly applied to the water fountain spigot.
(Appendix 1). The isolates were cultured on an R2A agar plate. Plates were reswabbed to
maintain isolate colonies (Appendix 2). Forty isolated colonies from the five fountains were
reswabbed on TSA plates and given designated isolate codes with the prefix WF for “water
fountain” (Table 1). The isolates were then selected based on their diversity in colony
morphology. Post restreaking, four new agar plates were prepared for identification (Figure 5).
MALDI-TOF MS
Isolates obtained were prepared for identification for MALDI-TOF MS following the Formic
Acid/Ethanol Tube Extraction Protocol [15]. First, the suspension was made using one hundred
percent ethanol. Seventy percent formic acid at a 1:1 ratio was then used to suspend the bacteria.
Acetonitrile was then added. The final supernatant was stored at -20℃ for target spotting.
Kirby Bauer Testing
Following identification, Polymyxin B, Chloramphenicol, and Penicillin were used in a Kirby
Bauer test for each isolate. To start, bacterial suspensions were then arranged by one millimeter
of TSB and a loop containing the pure culture was suspended into a test tube and then vortexed
thoroughly until the mixture was homogenous. A Mueller-Hinton agar plate was used to streak
the isolate suspensions. Four new agar plates that were marked with four quadrants were
prepped; each quadrant was designated for the three different antibiotics as well as a control
group. The antibiotic was set in its designated spot, sealed, labeled, and then incubated at 30℃
for twenty-four hours. After growth was allowed to happen the zone of clearance was measured
in millimeters, and was compared to known resistance and susceptibility values of the tested
antibiotics [12, 13]. The resistance and susceptibility values are listed below (Table 2).
Results (Madysson Volkman)
MALDI-TOF Identification and Analysis
Forty isolates were tested however only fifteen IDs were received. The WF06, WF13, and WF26
isolates were all Bacillus sp.. Brevundimonas sp. was seen in the WF10, WF20, and WF32
isolates. These are most likely different species. Pathogens found in the isolates include
Microbacterium sp, Eschericia coli, and Brevundimonas sp.(Table 3). From the cluster analysis
performed, there was not distinct clustering between the water fountains tested (Figure 7).
Table Three: MALDI-TOF results from isolate streaking.
WF04
Microbacterium sp.
WF05
Escherichia coli
WF06
Bacillus sp.
WF09
Blastomonas sp.
WF10
Brevundimonas sp.
WF12
Methylobacterium sp.
WF13
Bacillus sp.
WF19
Methylobacterium rhodesianum
WF20
Brevundimonas sp.
WF22
Sphingomonas sp.
WF26
Bacillus sp.
WF32
Brevundimonas sp.
WF33
Sphingomonas adhaesiva
WF34
Aquabacterium citratiphilum
WF35
Acidovorax delafieldii
Figure 7: MALDI-TOF Cluster Analysis of Five Water Fountains
Kirby Bauer Test
The isolates WF04, WF25, and WF32 were susceptible to penicillin. It was also observed that
WF06, WF07, WF20, and WF35 were resistant for Penicillin. Chloramphenicol susceptibility
was seen in WF04, WF06, WF20, WF26, and WF29. Isolate WF07 was Chloramphenicol
resistant. Polymyxin B susceptibility was seen in WF26, WF32, and WF35. Resistance to
Polymyxin B was seen in WF06, WF07, and WF20 were resistant. The only isolate to be
multi-drug resistant was WF07 (Table 4).
Comparison of a Similar Study
In a separate study, filtered versus unfiltered water was tested. The filtered water was derived
from automatic water bottle-filler fountains while the unfiltered water was derived from standard
water fountains. The standard water fountains are comparable to the ones tested in our study. The
MALDI-TOF results show clear clustering between the two water sources. Opportunistic
pathogens E. coli and Bacillus sp., and Brevundimonas sp. were present.
Figure 9: MALDI-TOF Cluster Analysis from a Separate Water Sample Study
Discussion (Aliyah Barreiro)
Based on the MALDI-TOF results, there was no distinct clustering between the five water
fountains tested. This suggests that the source of the bacterial similarities come from a single
source. The pipes, filter, or water supply are probable sources. In the isolate results, opportunistic
pathogens were present. These included E. Coli, Bacillus sp., and Brevundimonas sp. The lack of
clustering in the MALDI-TOF analysis suggests that the fountains could all contain these
pathogens. These pathogens could have originated from the fountains or from the water supplied
to the fountains. In a separate study, filtered and unfiltered water supply were tested. The
MALDI-TOF results show clustering between the filtered and unfiltered fountain bacterial
identities respectively. This suggests that there is no clear correlation between the filtered and
unfiltered water. Similar to our study, the pathogens E. coli, Bacillus sp., and Brevundimonas sp.
were found. An increased amount of E. coli was found in the filtered water system. Depending
on the strain, both E.coli and Bacillus sp. could be harmless gut bacteria or cause gastrointestinal
irritation like vomiting and diarrhea. Although classified as an opportunistic pathogen,
Brevundimonas sp. causes nosocomial infections and may not be harmful in non-clinical settings
[9]. Future isolate genetic testing could be performed to identify the strain and its pathogenic
potential.
If a person were to contract these pathogens and exhibit symptoms, it is important to know what
antibiotics can best treat the issue, and if the pathogens have acquired resistance. Antibiotic
resistance in the pathogens may prove treatment useless. The Kirby-Bauer antibiotic
susceptibility tests found resistance for Penicillin, Chloramphenicol, and Polymyxin B on several
water fountains. Many of the isolates were resistant to at least one antibiotic, and WF07 was
multidrug resistant. Some isolates did not grow at all on the media for the test. The species are
fastidious, which are unculturable on standard complete media [14]. Polymyxin B gave
surprising results. This antibiotic disrupts lipopolysaccharides (LPS) in Gram-negative cell
membranes. However, the antibiotic also inhibited Gram-positive bacteria. This is rare.
Polymyxin B has been shown to inhibit growth in Gram-positive bacteria not by altering the
membrane, but by disruption of secretion methods and/or an unknown internal target [10]. Both
the Kirby-Bauer and MALDI-TOF tests gave us results limited to the biota of the water fountain
surface. Comparing the results to the water from the fountains can give a more complete picture.
Acknowledgements
We would like to thank the University of Houston-Clear Lake for the opportunity to conduct this
research. There were no scholarships awarded in the funding of this project.
Appendices (Michael Nelson and Vernon Gradney)
Appendix 1: Cotton swabs were used for each water fountain to collect the samples of each of
the water fountains. Total of five swabs were used for the experiment.
Appendix 2: This plate grew bacteria from water fountain 32. Bacteria was grown on an
R2A-Agar plate
Appendix 3: The image also depicts the bacteria from water fountain 32. Bacteria was grown on
a TSA plate.
Appendix 4: The image includes swabbed water fountains 32-35 which were organized and all
grown on TSA plates
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