Short communication
Open Access
Bleach-tolerant Bacterial Species Isolated from
Potable Water in Hong Kong
Subramanya Rao1, Mui Kwok Wai2, Wong Ling Tim2 and Leung Polly1*
1Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
2Department of Building Services Engineering, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
*Corresponding author: Polly Leung, Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hung Hom, Kowloon,Hong Kong, China. E-mail:
@
Received: January 18, 2017; Accepted: February 05, 2017; Published: February 26, 2017
Citation: Subramanya Rao, Mui KW, Wong LT, Leung Polly (2016) Bleach-tolerant bacterial species isolated from potable water in Hong Kong. SOJ Microbiol Infect Dis 5(1): 1-4. DOI:
10.15226/sojmid/5/1/00164
Sodium hypochlorite (NaOCl) is a common household bleach. The
ability of this alkaline salt to kill a wide variety of harmful bacteria
upon contact makes it the chemical of choice in treating potable
drinking water. Several studies have shown the ineffectiveness of
the bleach in killing certain bacteria. We hypothesize that bleach
might not be effective to all type of bacteria, especially the one
which has the ability to form biofilms. Here we report the isolation
and phylogenetic identification of bleach-tolerant bacteria from
potable drinking water. The bacterial species isolated from this
study phylogenetically affiliated to Ralstonia picketti, usually found
in occurrence with biofilm-associated microflora.
Keywords: Biofilm; Disinfectant; Sodium hypochlorite; MALDI-TOF
MS; 16S rRNA gene
Introduction
Water is an essential element for life, contamination of tap
water as a result of improper water treatment and disposal
serves as a route of the transmission of water-borne diseases.
Therefore, provision of safe and clean potable water is one of the
most important tasks of the public health sectors.
Chlorine is the chemical of choice in treating potable drinking
water due to its effectiveness, high solubility and low cost [1,2].
When mixed with water, hypochlorous Acid (HOCl) is formed,
which is a strong oxidizing agent that kills bacteria and viruses
[3]. It has been documented that exposure to chlorine for about 1
hour achieved a great deal of reduction in the indicator bacterial
species in effluent samples [4]. However, this does not necessarily
applicable to the entire bacterial population in the water body
as the biofilm organisms are resistant to disinfectants [5]. In
our experiment, we aimed to test the effectiveness of NaOCl in
killing of potable water microorganisms, especially Legionella,
in an artificially constructed potable water distribution system.
Furthermore, we also attempted to isolate NaOCl-tolerant
bacterial strains. Here we report the isolation and phylogenetic
identification of bleach-tolerant bacterial genus Ralstonia from
potable drinking water.
Method
Construction of Model water systems
A model water distribution system was constructed in
order to investigate the ecological relationship between biofilm
microorganisms and Legionella in potable water distribution
system. Each system comprised an 30-L electric heater, which
connected to a potable water circulation system, a makeup water
supply system, a duplicate variable speed water pump set (with
one pump set served as the standby in case the duty pump was out
of order), and control accessories for monitoring temperature,
flow rates and other physicochemical properties of water.
In potable water circulation system, the speed pump fed water
through the water heater where the water temperature was
maintained at 25º C and the pump forced the water through the
system at sufficient pressure to ensure appropriate circulation
flow rate (0.3 ms-1).
Introduction of water with microbial consortium into
the model systems
Twenty litres (20L) of water collected from a water tap within
a university campus were fed to the model systems. As the water
sample was found to be negative for Legionella pneumophila
after screening, Legionella pneumophila (ATCC33152) was then
inoculated into the system at a concentration of 103 colonyforming
units (CFU)/ L, this concentration was based on the
findings of Legionella counts in potable water system [6]. After
feeding of water into the model systems, water was circulated
at a flow rate of 1.5 ms-1 for 8 hours in order to distribute the
microorganisms within the water systems. After that, water flow
rate was maintained at 0.3 ms-1 for 4 weeks.
Disinfection procedures
Ten 1-ml aliquots of water samples from the model water
system were obtained at the end of the 4th week. The samples
were placed in 1.5-ml tubes and were then treated with 0 ppm,
0.1 ppm, 0.5 ppm and 50 ppm of bleaching solution for 5, 10
and 30 minutes. One hundred microliters of water sample from
each tube were neutralized with 100 μl of sodium thiosulphate
(10% weight/volume). The neutralized sample was spread on
to Buffered Charcoal Yeast Extract (BCYE) agar plates (Oxoid
Microbiology products, UK), which is a selective growth media
used to isolate gram-negative bacterial species, particularly
Legionella pneumophila [7]. These plates were then incubated
at 37oC in 5% carbon dioxide for 3 days (2-5 days depended on
the growth of bacteria). After incubation, the bacterial colonies
were counted. Identities of the bacterial colonies were analyzed
using matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a new
technology for rapid identification of bacterial species based
on protein molecules [8, 9]. Bacterial identities were further
confirmed using PCR amplification and sequencing of the 16s
rRNA genes as described below.
DNA extraction, PCR, and sequencing
DNA was extracted from the bacterial isolates using
PowerWater culture DNA isolation kit according to the
manufacturer’s protocol (MO BIO Laboratories Inc., Carlsbad,
CA, USA). DNA amplification was performed by PCR. Forward
primer 27F (AGAGTTTGATCMTGGCTCAG) and reverse primer
907R (CCGTCAATTCMTTTRAGTTT) were used to amplify the
16s rRNA gene region [10]. The PCR profile included an initial
denaturation step at 95°C for 10 min, followed by 30 cycles of
amplification which consisted of denaturation at 95°C for 1 min,
primer annealing at 52°C for 50 s and extension at 72°C for 1
min. This was followed by a final extension at 72°C for 10 min.
The presence of PCR products was confirmed by electrophoresis
in 1% agarose gels. Purification of PCR product was carried out
using GFX PCR DNA and Gel Band Purification Kit (GE Healthcare,
UK). Automated DNA sequencing was performed using dyeterminator
cycle sequencing. After cycle sequencing, the purified
product was analyzed by capillary electrophoresis on a ABI3730
Genetic Analyzer (Applied Biosystems, US).
Phylogenetic analysis
The DNA sequences obtained were aligned with reference to
the selected GenBank sequences using ClustalW (http://www.
genome.jp/tools/clustalw/). Maximum likelihood analysis was
conducted using PAUP* 4.0b8 [11]. Phylogenetic trees were
drawn using Fig Tree. Bootstrap values (1,000 replications)
were shown for branch nodes supported by more than 70 %. All
sequences have been deposited in the NCBI GenBank database
under accession numbers (KX025085 - KX025096).
Results and Discussion
The results revealed that a total of 11 bacterial taxa comprised
the Ralstonia taxa. Ralstonia is usually associated with biofilms
in water samples. The strains grew on BCYE after treatment
with bleach solution at all three concentrations and at different
exposure durations. The total bacterial count for the untreated
water sample at T= 0 min was 3.5X105/L. At 0.1 ppm chlorine
concentration, the viable bacterial counts under 5, 10 and 30
min treatment duration were 8.0X104/L, 5.4X104/L, 4.9X104/L,
respectively. For the chlorine concentration at 0.5 ppm, the viable
bacterial counts under 5, 10 and 30 min treatment duration were
3.2X104/L, 4.1X104/L, 7.8X104/L, respectively. For the chlorine
concentration at 50 ppm, the bacterial counts under 5, 10 and
30 min treatment duration were 4.4X104/ L, 7.9X104/ L, 6X104/
L, respectively. According to the results, there was less than
10-fold reduction in bacterial counts under various treatment
conditions. Since the bacterial loads were still high even after 50
ppm NaOCl treatment for 30 min, we investigated the Identities
of the bacterial species that were resistant to NaOCl treatment.
Bacterial isolation using BCYE culture media yielded a total
of 11 isolates from 11 samples. These isolates were subjected
to MALDI-TOF MS-based identification and further confirmed
by 16S rRNA PCR and sequencing. Using MALDI-TOF MS, seven
isolates were identified as Ralstonia picketti and four bacterial
isolates were not identified (Table 1). Using 16S rRNA PCR and
sequencing, followed by BLAST search matching against the
GenBank database, all 11 bacterial isolates were identified to be
Ralstonia picketti or Ralstonia species (Table 1). Phylogenetic
analysis revealed that these cultivated isolates resistant to
disinfection were Ralstonia picketti isolates (Figure 1).
Table 1: Bacterial identities based on MALDI-TOF MS and 16S rDNA
sequencing.
Treatment condition (chlorine concentration/Time of exposure) |
Bacterial identity based on MALDI-TOF MS |
Bacterial identity based on 16S DNA sequencing and BLAST match (Accession number) |
BLAST Identity |
Accession number |
Nil/T=5 or 10mins |
Ralstonia picketti |
Ralstonia picketti strain CP12 (KF378754) |
99% |
KX025094 |
0.1ppm/T=5mins |
No Organisms Identified |
Ralstonia picketti strain CP12 (KF378754) |
99% |
KX025085 |
0.1ppm/T=10mins |
No Organisms Identified |
Ralstonia picketti isolate DiSca7 |
96% |
KX025086 |
0.1ppm/T=30mins |
Ralstonia picketti |
Ralstonia sp.PH-S1 (JN543508) |
99% |
KX025087 |
0.3ppm/T=5mins |
Ralstonia picketti |
Ralstonia picketti strain CP12 (KF378754) |
98% |
KX025088 |
0.3ppm/T=10mins |
No Organisms Identified |
Ralstonia sp.PH-S1 (JN543508) |
99% |
KX025089 |
0.3ppm/T=30mins |
No Organisms Identified |
Ralstonia picketti strain CP12 (KF378754) |
99% |
KX025090 |
50ppm/T=5mins |
Ralstonia picketti |
Ralstonia sp.PH-S1 (JN543508) |
99% |
KX025091 |
50ppm/T=10mins |
Ralstonia picketti |
Uncultured Ralstonia sp. Clone M_KL_110_14(KP967487) |
99% |
KX025092 |
50ppm/T=30mins |
Ralstonia picketti |
Ralstonia picketti strain CP12 (KF378754) |
99% |
KX025093 |

Figure 1: Phylogenetic relationships of cultivated NaClO tolerant strain
and related species based on 16s rRNA gene. Tree topologies are supported
by bootstrap values for 1,000 replications. Scale bar, 0.02-nt
change per position.
Ralstonia picketti is a Gram-negative bacterial species
frequently found in water and known for its ability to form
biofilms in aquatic environments [12,13]. Our findings showed
that the isolates could survive various NaOCl treatment
conditions, even at 50 ppm concentration for 30 minutes.
Earlier studies showed that Ralstonia pickettii had the ability
not only to survive but also to thrive in oligotrophic conditions
[12, 13] this might be attributed to its bio-degradative abilities.
Furthermore, the ability of Ralstonia picketti to form biofilms
[14] may help to resist to biocides and making Ralstonia difficult
to be eradicated even with the higher concentration of sodium
hypochlorite. Furthermore, Ralstonia pickettii has been also
recovered from a wide range of clinical environments and
emerged as an opportunistic pathogen [15]. Although the study
was designed to isolate Legionella pneumophila strains using
Legionella-specific growth medium. In this study, we were not
able to recover Legionella strain using culture-based methods
in both untreated and treated water samples, the reasons were
unknown. However, in our another study, < 1% Legionella was
detected in both untreated and treated potable water samples
using high throughput sequencing methods (Unpublished data).
Previous research has successfully demonstrated a 5-log
decrease of Legionella species in 5 hours using hyper chlorination
[16]. Our study has demonstrated that even after disinfection
using 50 ppm NaOCl for 30 minutes, we were able to isolate
Ralstonia picketti. This further reflects the ineffectiveness of
bleach in eradicating this bacterial species in potable water.
The possible reasons for bleach toleration in Ralstonia could
be due to its bio-degradative ability [13] and its ability to form
biofilms in potable water [14]. Recent studies demonstrated that
microorganisms survive within biofilm can acquire microbial
resistance to chemical disinfectants [17]. In summary, our
study has isolated a disinfectant-tolerant bacterial species from
chlorinated potable water system. It is likely that the oligotrophic
environment in water and the ability to utilize a variety of
compounds as carbon and nitrogen sources by this organism
facilitated its survival and propagation in such environment. A
detailed study on the entire bacterial genome would enhance
our current knowledge on the genes that associated with high
tolerance to NaOCl. Furthermore, it is also important to study the
relationship between Ralstonia and Legionella, as Ralstonia form
biofilm which favors the survival of Legionella.
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