Bio2 Lab – Literature Analysis ActivityTips for Reading Primary Literature (adapted from Wenk and Tronsky, 2011):
Most primary articles begin with an abstract, which summarizes the major points of the
study (what was asked, what was done, what was learned). If the subject is new to you, the
abstract may be hard to understand because it doesn’t explain much. It sometimes helps to
scan an abstract to see if the research is even close to what you want to know.
The introduction is usually helpful because it sets out the rationale for this study by telling
you four things:
• the general topic the paper addresses,
• previous work that led to the question asked in this study (citations to studies included in
the bibliography are given, but few details of that work are mentioned),
• the question(s) the study you are reading is designed to address, and
• the authors’ hypothesis(es).
The first time you read the paper, you might want to skip from the introduction to the
discussion to get a quick idea about what they conclude.
The methods section often has lots of technical details, so at first, focus on two things:
• An overall picture of the experimental design. Sometimes this information is set out more
clearly in the introduction or the abstract, but it’s important for you to step back from the
details and figure out why they designed the study as they did (more about this in the
handout on experimental design).
• Details about each step of the experiment (some of these—like how they chose their
subjects and how many subjects they studied and over what period of time— will be
important to understand right away; others have more detail than you need to worry
about).
The results section shows the results of tests described in the methods section. It shouldn’t
have much in the way of conclusions. What it will have are tables, graphs, or diagrams. The
text of the results discusses some of what is in those figures, but you’ll need to look closely
at the tables and graphs to really understand the results.
The next section is generally called discussion or conclusions. That’s where the authors
remind you of the original question(s) they were asking and address how well they think
their data answered those questions. They may refer to other studies that help explain some
of what they found or expected to find and didn’t. They may speculate in this section about
what their results might mean (including any alternative explanations) and what further
studies they believe need to be done and why.
The final section is the bibliography. This is very useful as you’re getting into a new topic. It
tells you who else is working in the field and what work was done earlier that led to this
study.
1
You will be reading and analyzing the following 15-page article (posted on Canvas):
Investigation into the microbial contamination in a spring water distribution system,
Western Cape, South Africa (Behardien et al., 2011;
https://academicjournals.org/article/article1380275357_Behardien%20et%20al.pdf )
Questions:
1. What section would you read if you wanted to quickly find out if a paper had
information that was useful to you? ___________________
2. Do the authors of this study state a hypothesis or research questions? If so, please
describe them.
Where did you find this information? ___________________
3. In 3-4 sentences, please describe why the authors did this study?
Where did you find this information? ___________________
4. Briefly describe the experimental design for this study. Do they have controls for
their study? If so, please describe.
5. Examine Figure 5. What conclusion can you draw from this graph? Make sure to first
look at the title and understand all abbreviations and your axes. Here are some
definitions to help you out with Figure 5:
A CFU or colony forming unit is a measurement of the number of culturable bacterial
cells in a given sample. Those individual cells give rise to visible colonies when plated
on an agar medium. These colonies are counted and incorporated into the following
equation in order to estimate the CFUs (we will be using this equation later in the
semester):
CFU/g =
or CFU/mL
(# of colonies x dilution factor)
volume of culture plated
2
6. Examine Figure 7.
a. What kind of diagram is displayed in this figure?
b. What are these types of diagrams used to show?
c. What experimental methods in the lab were done to enable the
bioinformatics (i.e. computer examination of DNA sequence analysis) used
to construct figure? (Hint: think about what you have done this semester in
Bio2 Lab for the River City Science project.)
3
African Journal of Microbiology Research Vol. 5(20), pp. 3200-3214, 30 September, 2011
Available online http://www.academicjournals.org/ajmr
DOI: 10.5897/AJMR11.183
ISSN 1996-0808 ©2011 Academic Journals
Full Length Research Paper
Investigation into the microbial contamination in a
spring water distribution system, Western Cape,
South Africa
Latiefa Behardien1, Arnelia Paulse2, Vanessa Jackson2, Sehaam Khan2 and Wesaal Khan3*
1
Department of Food Technology, Faculty of Applied Science, Cape Peninsula University of Technology, Cape Town,
8000, South Africa.
2
Department of Biomedical Sciences, Faculty of Health and Wellness Sciences,
Cape Peninsula University of Technology, Bellville, 7535, South Africa.
3
Department of Agricultural and Food Sciences, Faculty of Applied Science, Cape Peninsula University of Technology,
Cape Town, 8000, South Africa.
Accepted 15 July, 2011
The microbial contamination in a spring water distribution system in the Western Cape, South Africa
was investigated. Sampling at various points from the spring and throughout the bottling system
started in February and continued until November 2004. The number of culturable cells was determined
using the heterotrophic plate count (HPC) and total microbial counts were evaluated by flow cytometric
8
analysis (FCM). Heterotrophic plate counts in the final bottled water ranged from 1.34 x 10 cfu/ml (week
4
8
1) to 5 x 10 cfu/ml (week 46). In comparison, the total cell counts (FCM) ranged from 2.09 x 10
7
microorganisms/ml (week 1) to 5.70 x 10 microorganisms/ml (week 46).The higher FCM counts
indicated that the flow cytometry technique was able to detect viable but non-culturable organisms in
the water and was thus more reliable for the routine quantitative enumeration of microbial populations
in water samples. 16S ribosomal ribonucleic acid (rRNA) of the bacterial species present was amplified
with PCR and phylogenetic trees were constructed using the neighbour-joining algorithm. The
sequenced isolates from the various water samples belonged to the major groups Bacillus sp, and
Enterobacteriaceae and included Shigella boydii, Serratia sp., Enterobacter asburiae and Pseudomonas
sp.
Key words: Bacterial contamination, flow cytometry, heterotrophic plate count, molecular typing, spring water
distribution system.
INTRODUCTION
Natural spring water is obtained directly from underground water sources and is collected under conditions
that maintain its natural chemical composition and
microbiological purity. According to the South African
National Standards for Bottled Water (2003) the source of
*Corresponding author. E-mail: wesaalkhan5@gmail.com. Tel:
+27 21 460 3175. Fax: +27 21 460 3193.
the spring must not be situated at or close to any danger
of pollution by sewerage, farming operations, waste
disposal or industrial activities or any combination of the
above pollutant sources. Natural bottled water can also
only be subjected to certain treatment processes such as
the separation from unstable constituents by decantation
and or filtration, aeration, and by any process that will
ensure that the natural mineral content is not modified,
such as ultraviolet irradiation and ozonation (South Africa
Department of Health, 2004).
Behardien et al.
The microorganisms associated with spring water, which
is derived from a ground water source, can also generally
be related to the type of microbial pollutants in the soil
and the surrounding environment. It is thus essential to
assess the microbial contamination risk or the level of
pollution at the location of the spring (Leclerc and
Moreau, 2002). Coliform bacteria, when detected in
treated water supplies, could also be indicative of
inadequate treatment, or post treatment of the water
system (World Health Organisation, 1996). Coliforms are
present in large quantities in soil, and if found in water
they usually present a significant health risk. The water
source should then be routinely tested for faecal
contamination and coliforms are therefore used as
indicator organisms in bottled water analysis (Ryan,
2004).
Heterotrophic plate count bacteria are generally used to
assess the microbial quality of bottled water (SANS,
2003). In a random survey of bottled water conducted in
South Africa by Ehlers et al. (2004), heterotrophic plate
2
2
counts ranging from 1.1 x 10 to 5.4 x 10 cfu/ml were
recorded. It was concluded that the presence of these
high numbers were due to the natural microbial flora
present in the source water and could thus be used to
indicate the level of disinfection of the distribution and
bottling system (Leclerc and Moreau, 2002).
Flow cytometry is used to sort and measure different
types of cells by the fluorescent labelling of markers on
the surface of the cell (Javois, 1999). The addition of
fluorescent beads in conjunction with the Live/Dead
BacLight™ viability probe allows for the enumeration of
total bacteria in the water samples. Paulse et al. (2007)
assessed various enumeration techniques to investigate
the planktonic bacterial population in the Berg River,
Western Cape, South Africa. The heterotrophic plate
count technique was used to determine the number of
culturable microorganisms in the water samples and flow
cytometry was used to evaluate the total bacterial counts.
The study indicated that the average heterotrophic plate
count represented only a fraction (< 3.65%) of the total
flow cytometric analysis (FCM) counts and < 6.06% of the
viable FCM count.
Beuret et al. (2002) monitored three brands of natural
mineral waters in Europe over a one year period to
investigate and identify the microbial flora present.
Norovirus sequences were isolated from three leading
European brands of still mineral water, during the course
of this investigation. Research by Leclerc (2002) on the
microbiological safety of bottled water, identified the
major species of bacteria associated with natural mineral
water
as
Pseudomonas
fluorescent
species,
Pseudomonas non-fluorescent species, Acinetobacter,
Alcaligenes,
Comamonas
spp.,
Cyto-phaga
Flavobacterium, Arthrobacter and Corynebacterium.
The advantages of polymerase chain reaction (PCR) in
relation to other culture techniques or standard methods
3201
used for the detection of microbial pathogens in water, is
that it is specific, sensitive, rapid, accurate and can detect
small amounts of nucleic acids in a single sample. Tsen
et al. (1998) utilised PCR to select regions of the
Escherichia coli 16 S ribosomal ribonucleic acid (rRNA)
gene to detect these cells in water. The addition of an
enrichment step allowed for a detection limit of as low as
one E. coli cell /100 ml.
A spring water distribution system in the Western Cape,
South Africa experienced quality problems associated
with bacterial contamination. The aim of this study was to
investigate the bacterial contamination in this spring
water bottling system. The level of heterotrophic plate
counts (HPC) in the water samples at various sites
throughout the system were determined by the
conventional plate count technique. In addition, flow
cytometric analysis was used to obtain total cell counts
(the culturable and non-culturable populations) in the
collected water samples at the various sites. Identification of microorganisms in the water samples was
performed by means of molecular typing.
MATERIALS AND METHODS
Sampling sites
Sampling sites at the spring water distribution system in the
Western Cape, South Africa are indicated in Figure 1. The sites
include; Site A (borehole one); Site B (borehole two); Site C
(Dositron – Flushing point); Site D (between 0.3 µm filter and UV
steriliser –outside factory), Site E [After ultraviolet (UV)]; the bottling
line then splits into two lines and either one of the lines can be used
for bottling. Site F (Line one after 0.35µm filter); Site G (Line one
after 0.2 µm filter); Site H (Line two after 0.35 µm filter); Site I (Line
two after 0.2 µm filter) and Site J (at filler- final bottling point).
Sampling of these sites started in March 2004 (sampling batch one;
week one and sampling batch two; week four) and continued in
April (sampling batch three; week eight) until November 2004
(sampling batch four; week 46). Water samples were collected in 1
L sterile Nalgene-polypropylene bottles and stored on ice to
maintain a low temperature.
Heterotrophic plate count technique and pure culture isolation
Total heterotrophic plate counts were performed in duplicate on
R2A Agar (Merck, Biolab Diagnostics) after serial dilutions 10-1 to
10-7 of sample water was performed. Plates were incubated for 24
to 48 h at 37°C. Thereafter the number of visible cells, or colony
forming units (CFU’s) were counted and recorded. Distinct visible
cells CFU’s were identified and subcultured onto clean Nutrient
Agar (NA) (Merck, Biolab Diagnostics) plates for further purification
of cultures.
Flow cytometry (FCM)
The FCM outlined by Paulse et al. (2007) was employed in the
present study. Individual samples were subjected to a Becton
Dickinson FACSCalibur flow cytometer for analysis. The Becton
Dickinson FACSCalibur flow cytometer has a 15 mW, 488 nm
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Afr. J. Microbiol. Res.
Figure 1. Diagram of the borehole and bottling plant indicating sampling points.
argon-ion laser. A Doublet Discrimination Module, which uses pulse
width and area to eliminate cell clumping (doublets and triplets), in
conjunction with a LIVE/DEAD™ bacterial stain, allows for the
differentiation between bacterial cells and debris. An E. coli
laboratory strain was used as control.
Number of events in cell region
Number of events in bead region
X
Number of beads / test
Test volume
X
Dilution
(1)
[Bead concentration recorded at 988/µl for the Liquid Counting
Beads and at 49827 beads per Trucount™ tube, both obtained from
Biolab Diagnostics™]
DNA extraction and Agarose Gel electrophoresis
Cultures from planktonic samples obtained from the sampling sites
were spread-plated onto Nutrient Agar (NA) (Merck, Biolab
Diagnostics) after serial dilutions (10-1 to 10-7) of sample water were
performed. Plates were incubated for 3-4 days at 37ºC. Thereafter,
distinct visible cells [colony forming units (CFU)] were identified
based on morphological differences and re-streaked onto clean NA
plates for isolation of pure cultures. Deoxyribonucleic acid (DNA)
extraction was performed using the High Pure PCR Template
Preparation Kit as per manufacturer’s instructions (Roche
Diagnostics).
Extracted
DNA
samples
(10
µl)
were
electrophoretically analysed on a 0.8% molecular grade agarose
gel containing 12 µl of 0.5 µg/ml ethidium bromide, using 1 x Trisacetate- ethylenediamine tetraacetic acid (TAE) electrophoresis
buffer and run for one hour at 90 volts to confirm the presence of
genomic DNA.
Polymerase chain reaction (PCR)
The extracted DNA from individual samples was amplified using two
primer sets, respectively. Amplification of target DNA samples (5
µl) by PCR was performed in a total reaction volume of 50 µl
containing a 10 mM dNTP mix (1 µl), 25 mM MgCl2 (4 µl), 5 x PCR
Buffer with (NH4)2SO4 (10 µl), 10 µM forward (RW01) primer [AAC
Behardien et al.
3203
Figure 2. Average heterotrophic plate counts for the sites analysed recorded over the sampling period.
TGG AGG AAG GTG GGG AT] (2.5 µl), 10 µM reverse (DG74)
primer [AGG AGG TGA TCC AAC CGC A] (2.5 µl) (Greisen et al.,
1994), GoTaq DNA polymerase (0.25 µl) and sterile distilled H2O
(24.75 µl). For the second primer set all the reagents mentioned
above were added proportionally, together with 10 µM forward
(RDR080) primer [AAC TGG AGG AAG GTG GGG AC] (2.5 µl) and
10µM reverse (DG74) primer [AGG AGG TGA TCC AAC CGC A]
(2.5 µl) (Greisen et al., 1994) to obtain a total volume of 50 µl for
subsequent amplification. The PCR procedure included an initial
denaturation step of 5 min at 95°C, followed by 30 cycles of
amplification (25 s at 95°C, 25 s 55°C and 1 min at 72°C). The final
extension step was performed at 72°C for 10 min. Ten microliters of
the amplified DNA fragments of the PCR reactions were analysed
on a 1.2% agarose gel containing 12 µl of 0.5 µg/ml ethidium
bromide, using 1 x TAE electrophoresis buffer and run at 90 volts
for one hour to confirm successful amplification of the PCR product.
over the four sampling weeks were grouped and aligned with
ClustalX (1.81) using default parameters and the Blosum matrix. An
unrooted tree was constructed using the neighbour-joining (Saitou
and Nei, 1987) program of MEGA version 4.1 (Molecular
Evolutionary Genetics Analysis 4.1) (Tamura et al., 2007).
Branching patterns were evaluated by pairing 1000 replicates.
Statistical analysis
Repeated measures analysis of variance (ANOVA) [RMA] were
performed on all data obtained as outlined in Dunn and Clark
(1987) using StatisticaTM. In each RMA, the residuals were
analysed to determine if they were normally distributed. In all
hypothesis tests, a significant level of 5% was used as standards.
16S ribosomal RNA sequencing
RESULTS AND DISCUSSION
Successfully amplified PCR products (~400 kb) were purified using
a High Pure PCR Product Purification Kit as per the manufacturer’s
instructions (Roche Diagnostics). The DNA concentrations were
determined using the Qubit™ fluorometer (Invitrogen) and the
Quant-iT™ dsDNA BR (Broad-range) Assay kit 2–1000 ng as per
manufacturer’s instructions (Molecular probes and Invitrogen).
Samples were loaded onto 96-well plates (15 µl per sample), dried
in a speed vac with medium heat for 30 to 60 minutes (depending
on the volumes) and sent for subsequent sequencing where the
Applied Biosystems Big Dye Terminator v3.1 Cycle sequencing Kit
was used for the sequencing reactions, as per manufacturers’
protocols. Sequences were identified using the (Blastn) or Local
Alignment Search Tool Basic (Altschul et al., 1997) obtained from
the National Centre for Biotechnology Information website.
Heterotrophic plate counts (HPC)
Phylogenetic analysis
All the DNA sequences obtained from water at the various sites
Total culturable microbial counts obtained by the HPC
technique for all the sampling sites throughout the study
period are presented in Figure 2. The HPC recorded in
8
week one ranged from 1.34 x 10 cfu/ml at the borehole
7
(Site A) to 3.66 x 10 cfu/ml in the final bottled water (Site
8
J). The highest count of 2.02 x 10 cfu/ml was observed
after the 0.35 µm filter in line two (Site H) while the lowest
7
count of 3.0 x 10 cfu/ml was observed after ultraviolet
irradiation (Site E). The spring water system sampled
was experiencing problems with bacterial contamination
and the high counts could be ascribed to the fact that the
0.35 µm filter of line two was contaminated, clogged or
faulty or that a biofilm was present in the distribution
system which periodically sloughed off and served as a
3204
Afr. J. Microbiol. Res.
continuous source of contamination. In spring water
distribution systems however, filters must be backwashed
on a regular basis to maintain the integrity of the filter
system, as non-fixed pore filters enlarge in pore size after
high water volumes have passed through them, thus
resulting in the release of trapped contaminants into the
filtered water (Pall Filters, 2004). The lowest count
observed after UV indicated that the UV treatment was
effective in reducing the number of microorganisms,
however, the initial number of microorganisms in the
water was significantly high, and thus the UV irradiation
only reduced the microorganisms by one log cycle.
Senior and Dege (2005) confirmed that the efficiency of
UV irradiation as with any other disinfection process is
dependant on the quality of the incoming source water.
6
The HPC recorded in week four ranged from 4.50 x 10
6
cfu/ml at the borehole (Site A) to 9.00 x 10 cfu/ml in the
final bottled water (Site J). Increases in cfu/ml were
8
recorded after the 0.3 µm filter (Site D) at 3.10 x 10
cfu/ml and again after the 0.35 µm filter in line two (Site
8
H) at 2.39 x 10 cfu/ml. Blocked or contaminated filters
could have influenced the significant increase in HPC
counts recorded after these filters (Site D and H). A
significant decrease is however, noted after the water
passes through the UV system (Site E), line one at both
the 0.35 µm and 0.2 µm filters and the 0.2 µm filter in line
two (Site I). This indicates that these filters were still
functioning at their maximum efficiency. An increase in
the microbial count was recorded in the final bottled water
(Site J), which indicated that the filler ports were either
contaminated or had not been sanitised correctly. The
6
final microbial count of 9.0 x 10 cfu/ml (Site J) significantly exceeded the South African National Standard for
HPC for bottled water of < 100 organisms/ml.
The HPC recorded in week eight ranged from 2.70 x
8
7
10 cfu/ml at source (Site A) to 2.35 x 10 cfu/ml in the
final bottled water (Site J). The highest count was
recorded at the borehole (Site A) indicating that the
source water still contained significantly high numbers of
microorganisms, which would influence the filtration
process and the HPC count in the final bottled water. The
HPC count in the final bottled water (Site J) in week eight
7
was 2.35 x 10 cfu/ml in comparison to week one and
7
6
four where counts of 3.66 x 10 cfu/ml and 9.00 x 10
cfu/ml were recorded, respectively. The distribution
system was undergoing chlorine and oxonia disinfection
treatment processes to reduce microbial counts, but the
disinfection process was still not adequate to reduce the
number of microorganisms to the acceptable level of <
100 microorganisms/ml in the final bottled water. This
implied that based on the results for weeks one, four and
eight the spring water distribution system investigated,
required additional chlorine disinfections washes and the
filters had to be sanitised, backwashed or replaced to
achieve the legal requirement.
5
The HPC recorded in week 46 ranged from 4.5 x 10
4
cfu/ml at source (Site A) to 5.0 x 10 cfu/ml in the final
bottled water (Site J). The lowest HPC count was
recorded in the final bottled water while the highest
5
microbial count of 7.5 x 10 cfu/ml was observed after UV
irradiation (Site E). In week 46 a significant reduction in
HPC in comparison to week one, four and eight was
recorded for all sites as the system had been routinely
disinfected with chlorine soaks and oxonia. Contact times
and dosage for disinfecting the system were increased
and the filters were disinfected and backwashed.
However, the HPC count in the final bottled water still
significantly (p < 0.05) exceeded the acceptable limit of
< 100 organisms/ml.
Flow cytometric analysis (FCM)
The total cell counts obtained by flow cytometric analysis
are presented in Figures 3 and 4. The total cell counts
8
obtained in week one ranged from 2.09 x 10 micro7
organisms/ml at the large borehole (Site A) to 5.44 x 10
microorganisms/ml in the final bottled product (Site J). In
addition, significantly high total cell counts were observed
8
at the small borehole (Site B) at 2.03 x 10 micro8
organisms/ml, Dositron (Site C) at 1.88 x 10
microorganisms/ml, and after the 0.35 µm filter in line one
8
(Site F) at 1.56 x 10 microorganisms/ ml. The marked
increase in the total cell count observed after the 0.35 µm
filter in line one (Site F) indicated that the filter was
blocked and required sanitisation, backwashing or
replacement. A decrease in the total cell counts at Site D
7
7
at 6.13 x 10 microorganisms/m , Site G at 2.58 x 10
7
microorganisms/ml, and Site I at 2.11 x 10
microorganisms/ml, can be observed which indicated that
the filter system at these sites successfully retained some
of the bacterial load and supported the implementation of
multiple filter systems in a spring water distribution
system.
The total cell counts in week four ranged from
7
3.90 x 10 microorganisms/ml at the large borehole (Site
7
A) to 8.36 x 10 microorganisms/ml in the final bottled
product (Site J), with the lowest total cell count observed
7
at the Dositron unit (Site C) at 2.13 x 10 microorganisms/ml. The total cell count for Site F in week four
could not be measured as the sample vial broke. The
high total counts in the final bottled water in week four
indicated that although the bottling system was sanitised
with chlorine and oxonia, an increase in the cell count
was recorded from source to final product, which implied
that there was definitely a source of contamination in the
distribution system.
Tchobanoglous and Schroeder (1985) indicated that
the factors which influence the disinfection efficiency of
chlorine include the initial contact time, concentration and
form, microbial load, pH and temperature. The bottling
system of the site investigated was dosed with
Behardien et al.
3205
6E8
Total Cell Count/mL
5E8
4E8
3E8
2E8
1E8
WEEK 1
0
WEEK 4
WEEK 8
-1E8
A
B
C
D
E
F
G
H
I
J
WEEK 46
SITES
Figure 3. Enumeration of total bacteria by means of flow cytometric analysis (FCM) recorder over the sampling period.
Figure 4a. Dotplots of planktonic samples obtained at the
borehole for week 46 of the distribution site in the Western
Cape by means of Flow cytometric analyses (FCM).
concentrated chlorine and left to stand for two days to
increase its contact time. The initial counts in the source
7
water in week four of 3.90 x 10 microorganisms/ml and
7
3.88 x 10 microorganisms/ml at Sites A and B,
Figure 4b. Dotplots of planktonic samples obtained in the
final bottled water for week 46 of the distribution site in the
Western Cape by means of Flow cytometric analyses (FCM).
respectively were however, lower than the microbial
8
counts recorded at source in week one at 2.09 x 10
8
microorganisms/ml
(Site
A)
and
2.03 x 10
microorganisms/ml (Site B). The results indicate that the
precautions implemented to secure the borehole sites
such as the tapping of the boreholes, improved the
3206
Afr. J. Microbiol. Res.
Figure 5. Comparison of heterotrophic plate count to viable FCM count for week 46 for the sites analysed.
source water quality.
The total cell counts in week eight ranged from
7
7
6.25 x 10 microorganisms/ml (Site A) to 9.09 x 10
microorganisms/ml (Site J). The highest total cell count of
8
2.02 x 10 microorganisms/ml was recorded after UV
irradiation (Site E). For a UV light to function optimally the
quartz must be cleaned regularly to ensure full
transmissivity and efficacy (Senior and Dege, 2005).
Sommer and Cabaj (1993) evaluated the efficiency of a
UV plant for the disinfection of drinking water and
concluded that biodosimetric conditions should be used
to monitor disinfection efficiency. High counts of
8
1.80 x 10 microorganisms/ml at the Dositron (Site C) and
8
1.69 x 10 microorganisms/ml after the 0.2 µm filter line
two (Site I) were also observed, which indicated that the
dositron (point where sanitiser is added to the system)
was perhaps not sealed or was exposed to an external
source of contamination. Membrane fouling which is
caused by the accumulation of chemicals, particles and
growth of microorganisms on the membrane surface
could also have contributed to the increase in microbial
counts (Guidelines for Canadian Water Quality, 2008).
Due to the consistent contamination experienced, the
production at the supplier was stopped and the problem
was investigated. Sampling was resumed three months
later to measure the efficiency of the treatment
procedures implemented. The total cell counts recorded
8
in week 46 ranged from 2.69 x 10 microorganisms/ml at
7
the large borehole (Site A) to 5.70 x 10 microorganisms/
ml in the final bottled product (Site J). Results in week 46
8
fluctuated with the highest total cell counts of 5 x 10
8
microorganisms/ml and 5.08 x 10 microorganisms/ml
observed after UV irradiation treatment (Site E) and the
0.2 µm filter in line one (Site G), respectively. The high
total cell count recorded after the UV irradiation treatment
in week 46, compared to week one, four and eight
indicated the reduction in the efficiency of the quartz and
clearly showed that the lamp needed replacement.
In comparison to the other sampling weeks, low HPC
counts were observed in week 46, which indicated that
even though the counts still exceeded the stipulated HPC
limit, the treatment procedures implemented were
effective in reducing the CFU counts. However, the flow
cytometry results showed that the total cell counts of
week 46 were higher than all the other weeks sampled,
which clearly indicates that the HPC count was not a true
reflection of the microbial numbers in the spring water
distribution system.
The heterotrophic plate counts were thus compared to
the viable cell counts as obtained by flow cytometry. The
results for week 46 only (Figure 5) are discussed as a
representation of results as significant differences were
recorded in the HPC and FCM counts for this week.
These results showed that the flow cytometric (FCM)
analysis yielded higher viable counts in the water
sampled at the various sites. The highest CFU count of
5
7.50 x 10 microorganisms/ml was recorded after the UV
irradiation process (Site E). A corresponding FCM viable
6
count of 2.17 x 10 microorganisms/ml was recorded for
the same sampling site. The highest viable FCM count for
week 46 was observed at the Dositron (Site C) at 4.40 x
7
10 microorganisms/ml. A corresponding CFU count of
5
3.00 x 10 microorganism s/ml was recorded for the same
sampling site. The current water legislation, states that
Behardien et al.
3207
Figure 6a. Polymerase Chain Reaction analysis of extracted DNA samples (BB1-138 to 155) [with primer set 1:
forward (RW01) primer; reverse (DG74) primer] for sampling week 4. Lanes 1 –18: samples 14 to 32; Lane A:
Marker [MassRuler™ DNA Ladder Mix, #SM0403 (Fermentas)]; Lane B: Negative control.
Figure 6b. Polymerase Chain Reaction analysis of extracted DNA samples (BB1-138 to 155) [with primer set 2: forward (RW080)
primer; reverse (DG74) primer] for sampling week 4. Lanes 1 –18: samples 14 to 32; Lane A: Marker [MassRuler™ DNA Ladder Mix,
#SM0403 (Fermentas)]; Lane B: Negative control.
the heterotrophic plate count of the final bottled water
must be < 100 organisms/ml within 24 hours of bottling
however, no stipulation regulation for FCM in bottled
water could be found. Results clearly showed that in
comparison to the FCM technique, the heterotrophic plate
count technique, only allows for growth of the viable and
culturable cells present in the water samples and that it is
not an accurate method to assess the actual viable
microbial population in the bottled water samples (Paulse
et al., 2007).
Phylogenetic analysis
Figure 6 (a) and (b) represents the purified PCR agarose
gel electrophoresis photos of week 4 using both primer
sets 1 and 2. Lane one contains the DNA ladder #
SM0402 and lane two contains the negative control.
Phylogeny of 180 sequences were analysed and there
were many similar species that were repeatedly isolated
from the various sampling points over the sampling
periods. Their duplicate species were excluded as shown
in Figures 7 to 10. Species that were similar and
belonged to the same family were grouped together to
form clades. Bootstrap values for all scores were on
average above 90. Tables 1 to 4 indicate the different
bacterial species isolated from the various sites and their
GenBank accession numbers.
In week one, 16 diverse species were isolated and a
phylogenetic tree was constructed (Figure 7). Amongst
the organisms isolated were Shigella boydii, Serratia sp.
SB, Enterobacter asburiae, all of which belong to the
family Enterobacteriaceae.
Serratia belongs to the
coliform group and occurs in the environment, where their
presence usually indicates faecal contamination. In
developing countries contaminated drinking water is still
3208
Afr. J. Microbiol. Res.
Figure 7. Phylogenetic tree inferred from 16S rRNA sequence data, isolated from bacterial samples obtained
from water samples taken in week 1 from the distribution system in Western Cape, South Africa. Distance
matrices were constructed from the aligned sequences and created for multiple base changes at single position
by the BLOSUM algorithm.
also a major cause of shigellosis (Ray, 2004).
Pseudomonas and Stenotrophomonas sp. have also
frequently been isolated from mineral water (Leclerc,
2002).
In week four, 17 diverse species were isolated and a
phylogenetic tree was constructed (Figure 8). All the
species in this sampling week with the exception of
Pseudomonas sp. and Bacillus sp. were introduced in
this sampling period. In week four the highest HPC
counts were recorded at the filters (Figure 2) and it could
be at these contamination points where these species
were introduced. The presence of the Pseudomonas sp.
and Bacillus also indicated that the sanitisation process,
although adjusted was still not effective in the elimination
of these organisms from the water.
In week eight, 41 diverse species were isolated and a
phylogenetic tree was constructed (Figure 9). The highest
HPC count for all the sampling periods was recorded in
Behardien et al.
3209
Figure 8. Phylogenetic tree inferred from 16S rRNA sequence data, isolated from bacterial samples obtained
from water samples taken in week 4 from the distribution system in Western Cape, South Africa. Distance
matrices were constructed from the aligned sequences and created for multiple base changes at single position
by the BLOSUM algorithm.
week eight at the large borehole (Figure 2) and clearly
indicated the most pronounced point of contamination
and species introduction. The species introduced in this
sampling period included Commamonas aquatica,
Proteus sp. K10, 7 Proteus mirabilis, Hafnia alvei CCUG
429, Enterobacter sp. NJ-64, Enterobacter sp. MB-1-6-6,
Amorphomonas oryzae B46, amongst others.
The
presence of Pseudomonas sp. and Bacillus sp. again
indicate that these organisms were not eliminated during
the sanitisation process. The sanitisation process and
conditions were either insufficient to eliminate these
organisms or they could have formed a biofilm within the
3210
Afr. J. Microbiol. Res.
Figure 9. Phylogenetic tree inferred from 16S rRNA sequence data, isolated from bacterial samples obtained from water
samples taken in week 8 from the distribution system in Western Cape, South Africa. Distance matrices were constructed
from the aligned sequences and created for multiple base changes at single position by the BLOSUM algorithm.
distribution system.
In week 46, only 13 different species were isolated and
a phylogenetic tree was constructed (Figure 10). The low
species diversity correlates with the HPC count observed
in week 46, which was the lowest count for all the
sampling periods (Figure 2). The presence of
Pseudomonas sp. and Bacillus sp. again indicate that
these species were not eliminated during the sanitisation
Behardien et al.
3211
Figure 10. 16S rRNA sequence data, isolated from bacterial samples obtained from water samples taken in week 46 from the
distribution system in Western Cape, South Africa. Distance matrices were constructed from the aligned sequences and created for
multiple base changes at single position by the BLOSUM algorithm.
process and their persistence definitely indicates a biofilm
in the system. A study conducted by Percival et al. (1998)
on the development of biofilms on stainless steel pipes in
a mains water system indicated that the dominant specie
isolated were Pseudomonas spp. and Alcaligenes sp. In
week 46, the following species Escherichia sp.
Aeromonas, Endophytic bacteria and Brevundimonas sp.
were introduced. Escherichia sp. B4 belong to the family
Enterobacteriaceae and certain strains cause foodborne
gastroenteritis. The presence of Escherichia coli also
usually indicates faecal contamination (Ray, 2004).
The microbial flora isolated over the sampling period
was
mainly
Pseudomonas
sp.,
Bacillus
sp.,
Staphylococcus sp. and Stenotrophomonas sp.
Pathogens isolated over the sampling period include
Pseudomonas sp., Shigella, and Staphyloccocus sp. It is
thus important to understand quantitatively the bacterial
diversity in the bottling water distribution system in order
to apply and optimise the correct sanitisation procedure.
As bottled water cannot be subjected to any chemical
treatments during and after bottling it is important to apply
effective sanitisation, manage the filter integrity, UV
3212
Afr. J. Microbiol. Res.
Table 1. Table of 16 isolates, their codes and accession numbers for organisms isolated from week 1 sampling period.
Name presented on tree
75d Uncultured Pseudomonas sp.
CMG586 Pseudomonas aeruginosa
NBRAJG91 P. aeruginosa
WW5 Pseudomonas sp
CMG860 Pseudomonas aeruginosa
Pseudomonas sp.
Beta proteobacterium NOS8
Shigella boydii
Serratia sp. SB
E877 Enterobacter asburiae
Sphingomonas sp. ECN-2008
Bacillus sp. PK-7
JS-12 Bacillus sp
ISSDS-774 S. maltophilia
Stenotrophomonas sp.
R551-3 S. maltophilia
Organism
Clone_75d_Uncultured_Pseudomonas sp.
Pseudomonas aeruginosa strain CMG586
Pseudomonas aeruginosa strain NBRAJG91
Pseudomonas sp. WW5
Pseudomonas aeruginosa strain CMG860
Pseudomonas sp.
Beta proteobacterium NOS8
Shigella boydii
Serratia sp. SB
Enterobacter asburiae strain E877
Sphingomonas sp. ECN-2008
Bacillus sp. PK-7
Bacillus sp. JS-12
Stenotrophomonas maltophilia strain ISSDS-774
Stenotrophomonas sp.
Stenotrophomonas maltophilia R551-3
Accession number
EF593077.1
EU194236.1
EU661707.1
EF433547.1
EF511771.1
EF426444.1
AB076846.1
AB273731.1
EU816383.1
EF059885.1
AM940945.1
EU685824.1
EF040535.1
EF620464.1
AJ884482.1
CP001111.1
Table 2. Table of 17 isolates, their codes and accession numbers for organisms isolated from week 4 sampling period.
Name presented on tree
PK-7 Bacillus sp
ZZ2 Bacillus sp
PSA38 Bacillus cereus
CM24 Bacillus cereus
DB-10Bacillus sp
NA Bacillus subtilis
CT13 Bacillus pumilus
NBRAJATR9 Bacillus subtilis
AU55 Bacillus subtilis str.
MZ-32 Bacillus subtilis
STM 4035 Agrobacterium s.p
RRLJ SMAR Pseudomonas beteli
CL11b Endophytic bacterium
zf-IRht15 Acinetobacter sp
H5 Pseudomonas sp
ATCC 12633 Pseudomonas putida
NBRAJG91 P. aeruginosa strain
Organism
Bacillus sp. PK-7
Bacillus sp. ZZ2
Bacillus cereus strain PSA38
Bacillus cereus strain CM24
Bacillus sp. DB-10
Bacillus subtilis strain NA
Bacillus pumilus strain CT13
Bacillus subtilis strain NBRAJATR9
Bacillus subtilis strain AU55
Bacillus subtilis subsp. subtilis MZ-32
Agrobacterium sp. STM 4035
Pseudomonas beteli strain RRLJ SMAR
Endophytic bacterium CL11b
Acinetobacter sp. zf-IRht15
Pseudomonas sp. H5
Pseudomonas putida strain ATCC 12633
Pseudomonas aeruginosa strain NBRAJG91
irradiation and secure the spring source in order to supply
safe water free of any contamination.
Conclusions
The major conclusions of the study were as follows:
1. The lowest HPC count was recorded in week 46 but
Accession number
EU685824.1
DQ113449.1
EU346663.1
EU660318.
EU439408.1
EF064205.1
EU660365.1
EU661710.1
EF032684.1
EF422864.1
EF152474.1
DQ299947.1
EU088087.1
DQ223660.1
DQ268826.1
AF094736.1
EU661707.1
still notably exceeded the maximum limit of < 100
microorganisms/ml (South African National Standards for
Bottled Water, 2003).
2. The total cell counts obtained by the FCM method
were higher in week 46 at all the sites throughout the
period, when compared to the heterotrophic plate counts.
3. The higher FCM counts indicated that the flow
cytometry technique was able to detect cells in the
sample that enter a viable but non-culturable state and
Behardien et al.
3213
Table 3. Table of 43 isolates, their codes and accession numbers for organisms isolated from week 8 sampling period.
Name presented on tree
NBRAJG91 P. aeruginosa
NBRAJG91 P. aeruginosa
WW5 Pseudomonas sp.
CMG586 Pseudomonas aeruginosa
418 Pseudomonas sp
MCCB Pseudomonas aeruginosa
IL1 Pseudomonas aeruginosa
8.2 Pseudomonas sp
Pseudomonas aeruginosa
634 Comamonas aquatica
K107 Proteus sp
Proteus mirabilis
Hafnia alvei CCUG 429
NJ-64 Enterobacter sp
MB-1-6-6 Enterobacter sp.
45 Stenotrophomonas sp
R551-3 Stenotrophomonas maltop.
7-3 Stenotrophomonas sp.
Amorphomonas oryzae B46
TG8 Uncultured soil bacterium
Rhizobium soli strain DS-42
NASA2-43 Brevibacterium sp.
Arthrobacter sp. W17
SS-08 Staphylococcus pasteuri
AT2 Staphylococcus epidermidis
SA6 Staphylococcus sp
TG8 Uncultured soil bacterium
BMP-1 Bacillus sp.
Bacillus sp. X5
WS7b Endophytic bacterium
Gamma proteobacterium BDA453
S.arcachonense sp. nov.
HNR07 Bacillus sp.
770 Bacillus cereus
SS-07 Bacillus cereus
BGSC 6A16 Bacillus cereus
PSA38 Bacillus cereus
PK-2 Bacillus sp
ZZ2 Bacillus sp.
SCB001 Bacillus cereus
Organism
Pseudomonas aeruginosa strain NBRAJG91
Pseudomonas aeruginosa strain NBRAJG91
Pseudomonas sp. WW5
Pseudomonas strain CMG586
Pseudomonas sp. 418
Pseudomonas aeruginosa isolate MCCB
Pseudomonas aeruginosa isolate IL1
Pseudomonas sp. 8.2
Pseudomonas aeruginosa
Comamonas aquatica strain 634
Proteus sp. K107
Proteus mirabilis
Hafnia alvei CCUG 429
Enterobacter sp. NJ-64
Enterobacter sp. MB-1-6-6
Stenotrophomonas sp. 45
Stenotrophomonas maltophilia R551-3
Stenotrophomonas sp. 7-3
Amorphomonas oryzae B46
Uncultured soil bacterium clone TG8
Rhizobium soli strain DS-42
Brevibacterium sp. NASA2-43
Arthrobacter sp. W17
Staphylococcus pasteuri strain SS-08
Staphylococcus epidermidis strain AT2
Staphylococcus sp. SA6
Uncultured soil bacterium clone TG8
Bacillus sp. BMP-1
Bacillus sp. X5
Endophytic bacterium WS7b
Gamma proteobacterium BDA453
S. arcachonense sp. nov.
Bacillus sp. HNR07
Bacillus cereus strain 770
Bacillus cereus strain SS-07
Bacillus cereus strain BGSC 6A16
Bacillus cereus strain PSA38
Bacillus sp. PK-2
Bacillus sp. ZZ2
Bacillus cereus strain SCB001
that the heterotrophic plate count technique only allowed
for growth of the viable and culturable cells present in the
water samples.
4. Flow cytometry proved to be a rapid and more reliable
technique for the assessment of total bacterial count in
water samples.
5. The dominant species Pseudomonas sp. and Bacillus
Accession number
EU661707.1
EU661707.1
EF433547.1
EU194236.1
EU841539.1
EF053508.2
DQ989211.2
EF426444.1
EU327890.1
EU841530.1
EU710747.1
DQ777867.1
FM179944.1
AM421983.1
EU816586.1
AY856845.1
CP001111.1
EU054384.1
AB233493.1
DQ297948.2
EF363715.1
EU029632.1
EU596424.1
EU624447.1
EU021221.2
AY864655.1
DQ297948.2
DQ371431.1
EU236728.1
EU088038.
AB304258.1
Y11561.1
EU373351.1
EU430093.1
EU624445.1
AY310302.1
EU346663.1
EU685821.1
DQ113449.1
DQ466089.1
sp. were isolated throughout the sampling period from
week one to week 46. The pathogenic organisms isolated
from all the sampling periods included Escherichia sp.,
Pseudomonas sp., Shigella boydii, Bacillus sp. and
Staphyloccocus sp.
6. It is thus important to understand quantitatively the
viable bacterial load and the species diversity in the
3214
Afr. J. Microbiol. Res.
Table 4. Table of 13 isolates, their codes and accession numbers for organisms isolated from week 46 sampling period.
Name presented on tree
ZH4 Bacillus sp
BGSC Bacillus cereus strain
PSA38 Bacillus cereus
CT13 Bacillus pumilus
S8-07 Bacillus pumilus
JS-12 Bacillus sp
YIM KMY42-2 Brevundimonas sp
B4 Escherichia sp
WW7 Aeromonas sp
CN015 Pseudomonas putida
NBRAJG91 P. aeruginosa
PR Pseudomonas sp
CL13A Endophytic bacterium
Organism
Bacillus sp. ZH4
Bacillus cereus strain BGSC
Bacillus cereus strain PSA38
Bacillus pumilus strain CT13
Bacillus pumilus strain S8-07
Bacillus sp. JS-12
Brevundimonas sp. YIM KMY42-2
Escherichia sp. B4
Aeromonas sp. WW7
Pseudomonas putida strain CN015
Pseudomonas aeruginosa strain NBRAJG91
Pseudomonas sp. PR
Endophytic bacterium CL13A
bottling water distribution system in order to apply and
optimize the most efficient sanitization procedure.
7. As bottled water cannot be subjected to any chemical
treatments during and after bottling it is also important to
understand the survival capacity of the pathogenic and
indicator organisms.
ACKNOWLEDGEMENTS
The National Research Foundation (NRF) and Cape
Peninsula University of Technology (CPUT) for financial
support.
Abbreviations: HPC, Heterotrophic plate count; FCM, flow
cytometric analysis; rRNA, ribosomal ribonucleic acid; PCR,
polymerase chain reaction; UV, ultraviolet; CFU’s, colony
forming units; TAE, tris-acetate- ethylenediamine tetraacetic
acid; ANOVA, analysis of variance; RMA, repeated measures
ANOVA.
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EU088094.1
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