Effect of disinfectants on adhered Aeromonas hydrophila to polyethylene immersed in water under static and dynamic conditions

Abstract

The growth of A. hydrophila in liquid medium was monitored a hyperbola of four stages of growth. The effect of Sodium hypochlorite (NaOCl) (0.5, 1 and 1.5 ‰) and Hydrogen Peroxide (H 2 O 2) (5, 10 and 15 ‰) on the adherent cells under static and dynamic conditions on fragments of polyethylene immersed in water was assessed. Cells were harvested from different growth stages. With cells harvested from each growth stage, abundances of adhered A. hydrophila were generally lower in the presence of NaOCl and H 2 O 2 than in their absence. With the 2 disinfectants, a significant difference amongst the average densities of adhered A. hydrophila at each growth phase was observed (P<0.05). The effectiveness of each disinfectant concentration on adhered A. hydrophila decreased as the duration of the adhesion increased. Although the adsorption coefficient obtained from Freundlich isotherms was relatively higher in the static than in the dynamic regime, no significant difference was observed between the mean abundances of A. hydrophila adhered under these two experimental conditions (P>0.05). NaOCl seems more effective on A. hydrophila adhered to polyethylene than H 2 O 2. Adhered A. hydrophila to polyethylene under the dynamic condition was more sensitive to both disinfectants than that adhered under static condition. These results suggested that the incubation duration and the cell growth stage played an important role in the bacterial resistance mechanism towards disinfectants.

Full text

Int. J. Res. BioSciences 33
International Journal of Research in BioSciences
Vol. 2 Issue 2, pp. (33-48), April 2013
Available online at http://www.ijrbs.in
ISSN 2319-2844
Research Paper
Effect of disinfectants on adhered Aeromonas
hydrophila to polyethylene immersed in water
under static and dynamic conditions
Lontsi Djimeli C., *Nola M., Tamsa Arfao A., Nandjou Nguéfack R.V., Noah Ewoti O.V.,
Nougang M.E. and Moungang M.L.
Laboratory of General Biology, Hydrobiology and Environment Research Unit, Faculty of Sciences,
University of Yaoundé 1, P.O. Box 812, Yaoundé, Cameroon
(Received February 06, 2013, Accepted March 28, 2013)
Abstract
The growth of A. hydrophila in liquid medium was monitored a hyperbola of four stages of
growth. The effect of Sodium hypochlorite (NaOCl) (0.5, 1 and 1.5 ‰) and Hydrogen Peroxide
(H2O2) (5, 10 and 15 ‰) on the adherent cells under static and dynamic conditions on
fragments of polyethylene immersed in water was assessed. Cells were harvested from
different growth stages. With cells harvested from each growth stage, abundances of adhered
A. hydrophila were generally lower in the presence of NaOCl and H2O2 than in their absence.
With the 2 disinfectants, a significant difference amongst the average densities of adhered A.
hydrophila at each growth phase was observed (P<0.05). The effectiveness of each
disinfectant concentration on adhered A. hydrophila decreased as the duration of the
adhesion increased. Although the adsorption coefficient obtained from Freundlich isotherms
was relatively higher in the static than in the dynamic regime, no significant difference was
observed between the mean abundances of A. hydrophila adhered under these two
experimental conditions (P>0.05). NaOCl seems more effective on A. hydrophila adhered to
polyethylene than H2O2. Adhered A. hydrophila to polyethylene under the dynamic condition
was more sensitive to both disinfectants than that adhered under static condition. These
results suggested that the incubation duration and the cell growth stage played an important
role in the bacterial resistance mechanism towards disinfectants.
Keywords: adhered A. hydrophila, cell growth phase, effect, H2O2, NaOCl, water.
Introduction
One of the major concerns of companies in charge of the treatment of drinking water is to effectively
meet the demand and maintain the good quality of water in distribution [1]. The drinking water
distribution network is often the place of many physico-chemical and biological reactions resulting in
interactions between disinfectants, pipe walls, and free or fixed biomasses. These reactions are
sometimes the cause of the deterioration of the organoleptic properties of supplied water [2, 3].
Analysis of the water distribution quality is based on physico-chemical and microbiological parameters
[3, 1]. In recent years, the public health sector recognized A. hydrophila as an opportunistic pathogen,
implicated in gastroenteritis, septicemia, cellulitis, colitis, meningitis and respiratory infections [4, 5, 6].
To prevent bacterial re-growth a residual disinfectant is maintained in the water distribution network.
Ozone (O3), chlorine dioxide (ClO2), monochloramine (NH2Cl), free chlorine (Cl2), NaOCl, H2O2 are
disinfectants that can be sometimes used in water disinfection treatment [7].
Previous works have shown that A. hydrophila is a widespread species in the environment. This
microorganism has been isolated from lakes, rivers, sea water, sewage effluent, and especially in

Int. J. Res. BioSciences 34
water intended for human consumption [8, 9]. Its concentration is generally between 0 and 102 CFU/ml
in the outlet of drinking water treating plant. This concentration can be higher in drinking water
distribution networks due to its growth on biofilms [10, 8]. Ingestion of contaminated food or water is the
common route of advanced infection in the case of Aeromonas [11]. The pathogenicity of A. hydrophila
is expressed primarily in fish, seafood and amphibians [12]. With the humans, A. hydrophila is an
opportunistic pathogen that causes intestinal infections such as gastroenteritis [13] or extra-intestinal
infections such as cutaneous infections [14]. They are responsible for gastroenteritis and severe
diseases (septicemia, peritonitis ...), wound, genitourinary and ocular infections in immunodepressed
patients [15]. One number of virulence factors contributing to its pathogenicity. Besides enzymes
favoring infection (proteases, lipases, DNase), mobile Aeromonas have adhesion capabilities and
produce various toxins including endotoxins, enterotoxins, hemolysins and cytotoxins [16].
Many studies have also focused on monitoring, water supply, treatment plants, and the health risks
associated with the dysfunctioning of the distribution of drinking water [17, 18, 19]. They showed that
despite treatment done upstream added to maintain a disinfectant residual in pipelines and
oligotrophic medium, some bacteria adapt and proliferate in the water distribution network [1]. They are
sometimes the cause of nests and microbial biofilm formation among others. In addition, the variation
of microorganisms in response to disinfectants can be linked to changes in their cell wall which may
be due to a change in their growth stage [20]. While previous studies have allowed to understand the
mechanisms of emergence and evolution of biofilms of A. hydrophila in the drinking water distribution
system [8, 1], there is little information on the importance of the growth stages or metabolic process and
the reaction of biofilms of A. hydrophila against disinfectants. The present study aims at evaluating in
microcosm the effect of NaOCl and H2O2 on A. hydrophila bonded under different conditions at
different growth stages on polyethylene fragments immersed in water during time.
Materials and Methods
Collection and identification of A. hydrophila
The bacteria A. hydrophila was isolated from well water in Yaoundé (Cameroon) using membrane
filtration technique, on ampicillin-dextrin agar medium [21, 22]. Cell subculture was performed on
standard agar medium (Bio-Rad laboratories, France). The cells were then identified using standard
biochemical methods [23]. These cells are anaerobic facultative, non-sporulated, Gram-negative bacilli,
ferment mannitol, produce indole and mobile. They do not possess urease, lysine decarboxylase
(LDC), ornithine decarboxylase (ODC) and arginine dihydrolase (ADH). For the preparation of stocks
of bacteria, the Colony Forming Units (CFUs) from standard agar medium were inoculated into 100 ml
of nutrient broth (Oxford) for 24 hours at 37 °C. Afterwards, cells were harvested by centrifugation at
8000 rpm for 10 min at 10 °C and washed twice with NaCl (8.5 g/l) solution. The pellet was re-
suspended in NaCl (8.5 g/l) solution and then transferred to 300 µL tubes. The stocks were then
frozen stored.
Assessment of the cell growth phases
Three sets of 15 test tubes each containing 10 ml of sterile tryptone (Biokar) solution were used.
Tubes of each set were labeled t0, t2, t4, t6, t8, t10, t12, t14, t16, t18, t20, t22, t24, t26 and t28. Prior to the
experiments, the stock frozen vial containing cells was thawed at room temperature. The culture (300
μl) was then transferred into 10 ml of nutrient broth (Oxford) and incubated at 37 °C for 24 hours.
After, cells were then harvested by centrifugation at 8000 rpm for 10 min at 10 °C and washed twice
with sterile NaCl (8.5 g/l) solution. The pellet was then re-suspended in 10 ml of sterilized solution
containing NaCl (8.5 g/l) solution. After dilution, 100 µl was added to 100 ml of sterilized NaCl (8.5 g/l)
solution, in each of the 15 tubes containing sterilized peptone solution. Cell suspensions in the 3
tubes coded t0 were immediately analyzed. Those in tubes coded t2, t4, t6 … t28 were incubated for 2,
4, 6… 28 hours at 37 °C. The CFUs were counted after each incubation duration. The averages of the
CFUs were calculated from the results of the triplicates and the Log(CFU) also calculated. The
straight Log(number of CFUs) curve against storage duration was plotted and then assimilated as the
cell growth curve. The cell growth phases were then assessed.
Disinfectants and adsorbant substrates used
Two disinfectants were used: NaOCl, which belongs to the group of halogen derivatives and H2O2

Int. J. Res. BioSciences 35
belonging to the group of oxidants. The initial concentration of the H2O2 was 10 volumes. To count the
surviving bacteria after disinfection treatment, sterile NaCl solution (8.5 g/l) was used as diluent. The
disinfectant concentrations used ranged from 0.5 ‰ to 1.5 ‰ and from 5 ‰ to 15 ‰, for NaOCl and
H2O2 respectively. The easier use of these two disinfectants in the drinking water treatment has
justified their choice for this study.
The substrate used is high dense polyethylene. It differs from radical low dense polyethylene and
linear low dense polyethylene by the molecular structure of its sparsely branched chains, and its
relatively high resistance to shocks, high temperatures and ultraviolet [24, 25]. It is a plastic piping
material obtained directly from the supplier and used in drinking water distributing.
Adhesion protocol of A. hydrophila to polyethylene
On the basis of previous studies, parallelepiped fragments of polyethylene with 13.28 cm² of total
surface area suspended with inconsiderable diameter wire were immersed in triplicate in two sets A
and B each in four flasks 250 ml Duran A1, A1', A1'' and B1, B1', B1'', A2, A2', A2'' and B2, B2', B2'',
A3, A3', A3'' and B3, B3', B3'' and A4, A4', A4'' and B4, B4', B4'' each containing 99 ml of NaCl
solution (8.5 g/l). Meanwhile, the controls were made and were coded A01, A02, A03, A04 and B01,
B02, B03, B04 [26]. The whole was then autoclaved.
Prior to the experiments, stocks frozen vial containing A. hydrophila were thawed at room
temperature. Then 100 µl of the culture was transferred into test tubes containing 10 ml of nutrient
broth (Oxford) and incubated at 37 °C for 24 hours. Cells from a specific growth phase were then
harvested by centrifugation at 8000 rpm for 10 min at 10 °C and washed twice with sterile NaCl
solution (8.5 g/l). The pellets were then re-suspended in 50 ml of sterilized NaCl solution (8.5 g/l).
After serial dilutions, the concentration of bacteria in each solution was adjusted to 6x108 CFU/ml by
reading the optical density at 600 nm using a spectrophotometer (DR 2800) followed by culture on
agar [26]. 1 ml of the suspension was added to 99 ml of sterilized NaCl solution (8.5 g/l) contained in
an Erlenmeyer flask.
The flasks, A01, A1, A1', A1'', A02, A2, A2', A2'', A03, A3, A3', A3'', and A04, A4, A4', A4'' were
incubated under dynamic condition at a stirring speed 60 rev/min, using a stirrer (Rotatest brand). The
flasks, B01, B1, B1', B1'', B02, B2, B2', B2'', B03, B3, B3', B3'' and B04, B4, B4', B4'' were incubated
under static conditions. The incubation duration was 180 minutes for the Erlenmeyer flasks A01, A1,
A1', A1''and B01, B1, B1', B1''. That of A02, A2, A2', A2''and B02, B2, B2', B2'' was 360 min. For the
Erlenmeyer flasks coded A03, A3, A3', A3''and B03, B3, B3', B3'' and those coded A04, A4, A4', A4''
and B04, B4, B4', B4'', the incubation durations were 540 and 720 min respectively. All these
incubations were done at laboratory temperature (25 ± 1 °C).
Disinfection experiments
After each incubation duration, fragments of polyethylene were drained for 10 seconds in a sterile
environment created by the Bunsen burner flame and then introduced into test tubes containing 10 ml
of diluted disinfectant of various concentrations. Fragments removed from flasks A1, A2, A3, A4 were
introduced in disinfectant solutions of 0.5 ‰ NaOCl. Those removed from flasks B1, B2, B3 and B4
were introduced in 5 ‰ H2O2. Fragments removed from flasks A1', A2', A3', A4' and those removed
from B1', B2', B3' and B4' were introduced into 1 ‰ NaOCl and 10 ‰ H2O2 solutions respectively.
Similarly, those removed from flasks A1'', A2'', A3'', A4'', and from the flasks B1'', B2'', B3'' and B4''
were introduced into 1.5 ‰ NaOCl and 15 ‰ H2O2 solutions respectively.
Fragments of polyethylene flasks from A01, A02, A03, A04 and B01, B02, B03, B04 were introduced into
10 ml of sterile NaCl solution (8.5 g/l). After 30 min of incubation at room temperature and under static
conditions, each fragment was then drained out under sterile conditions. Each fragment was then
introduced into 10 ml of sterilized NaCl solution (8.5 g/l).The unhooking of adherent cells was
performed by vortex agitation at increasing speeds for 30 seconds in three consecutive series of 10
ml sterilized NaCl solution (8.5 g/l).This technique allows the unhooking of maximum adhered cells [27,
28]. The total volume of the suspension containing the unhooked bacterial cells was 30 ml. The
isolation and enumeration of unhooked cells was made by culture on ampicillin dextrin agar, by the
method of surface spreading, followed by incubation on Petri dishes at 37 °C for 24 hours.

Int. J. Res. BioSciences 36
Data analysis
The variation of the abundances of adhered A. hydrophila in each experimental condition was
illustrated by semi-logarithmic diagrams. Standard deviations were not fitted because the curves were
too close. Spearman "r" Test correlation was used to assess the degree of correlation between the
abundances of adhered A. hydrophila and other parameters considered. Kruskal-Wallis and Mann-
Whitney tests were used to compare the mean abundance of cells adhered from one experimental
condition to another.
Adhesion speeds of A. hydrophila on polyethylene were assessed constructing linear regression of
adhered A. hydrophila after each incubation duration of three hours by means of Excel program. The
ratio slope of the linear regression and three hours correspond to adhesion speeds of A. hydrophila
per hours. These results are expressed as adhered cells/cm²/hours.
The data from absorption experiments were analyzed using the Freundlich model. This isotherm was
chosen because of the number and the relevance of the information it provides on the real adsorption
mechanisms on one hand and its remarkable ability to match doses of adsorption on the other hand
[29, 30]. The Freundlich isotherm is described by the following equation [29, 30]:
Cs= Kf . Cl/n
With Cs; the quantity of cells adsorbed in the presence of disinfectant, C; the concentration of cells
adsorbed in the absence of disinfectant, Kf; the Freundlich coefficient adsorption which is connected
to the adsorption capacity, l/n; coefficient linearity, and n being the intensity of adsorption. Here, Cs is
expressed as number of adherent cells/disinfectant concentration and C, the number of adherent
cells/cm² of polyethylene. Constructing linear regression log Cs versus log C, resulting in a line of
slope l/n which intercepts the y-axis log Kf.
Results and Discussion
Cell growth curve
The growth of A. hydrophila in non-renewed peptone liquid medium explained a hyperbolic curve of 4
phases: a lag growth phase from 0 to 2 hours, an exponential growth phase from 2 to 13 hours, a
stationary growth phase from 13 to 22 hours, and a decline growth phase begins from the 22th hour
(Figure 1).
Figure 1: Growth curve of A. hydrophila (LP: Lag growth phase, EP: Exponential growth phase,
SP: Stationary growth phase, DP: Decline growth phase)

Int. J. Res. BioSciences 37
Adhesion kinetics in static and dynamic conditions
The hourly cell adhesion speeds on the considered substrate was assessed in static and dynamic
conditions. It varied from one growth phase to another. Under static condition, adhesion speeds
varied from 0.002 to 0.870 cell/h. Under dynamic condition, it varied from 0.005 to 0.930 cell/h. In both
conditions, the lowest cell adhesion speeds were registered with cells harvested from the stationary
growth phase whereas the highest were noted with cells harvested from the lag growth phase (Table
1).
Table1: Hourly adhesion speeds (and regression coefficient) of A. hydrophila with respect to
growth phases under static and dynamic conditions.
Abundance of A. hydrophila adhered to polyethylene after NaOCl disinfection
The densities of adhered A. hydrophila ranged from 0 to 256 CFU/cm² after the action of NaOCl. The
maximum abundance of adhered A. hydrophila was recorded under dynamic condition, in the
presence of 0.5 ‰ NaOCl and this after 720 min with cells harvested from the lag growth phase.
Adhered A. hydrophila was sometimes been totally decimated by NaOCl. This result was observed
during the decline phase in static and dynamic conditions in the presence of three concentrations of
NaOCl, at the end of all incubation durations. The same observation was made for adhered A.
hydrophila during the stationary growth phase, in the presence of 1.5 ‰ NaOCl at 180 and 360 min
incubation durations (Figure 2).
With cells coming from the lag phase, the abundance of A. hydrophila adhered to the control
substrate varied throughout from 105 to 1799 CFU/cm² and had always been superior to those of
fragments tested for disinfection. In addition, they increase with the incubation duration. Maximum cell
density was recorded after an adhesion test of 720 min under dynamic condition when studying the
impact of NaOCl. After action of NaOCl, the densities of adhered A. hydrophila ranged from 1 to 256
CFU/cm². The effectiveness of NaOCl decreased with the length of the adhesion duration test.
Maximum cell abundance was recorded in the presence of 0.5 ‰ NaOCl after an adhesion test of 720
min in dynamic regime. The lower density of adhered cells was observed in the presence of 1.5 ‰
NaOCl with cells coming from the adhesion tests of 180 min under static condition (Figure 2).
Abundances of A. hydrophila adhered to control substrate during the exponential growth phase were
also higher than those fragments tested for disinfection in the lag growth phase. They generally
fluctuated between 12 and 313 CFU/cm². After disinfection test, it was noted that the effectiveness of
NaOCl decreased when the duration of adhesion test increased. Abundance of adhered A. hydrophila
ranged between 3 and 59 CFU/cm² (Figure 2).
With cells coming from the stationary growth phase, abundance of A. hydrophila adhered to the
control substrate varied from 2 to 13 CFU/cm². They remained higher than those of fragments tested
for disinfection. After disinfection test, abundances of adhered A. hydrophila ranged between 0 and 3
CFU/cm². As the duration of adhesion test increased, it was noted that the effectiveness of NaOCl
decreased. The maximum density of A. hydrophila adhered to the polyethylene was recorded in the
presence of 0.5 ‰ NaOCl after 720 min incubation duration and under dynamic and static conditions.
The lower density of adhered cells was observed in the presence of 1.5 ‰ NaOCl after 180 and 360
Cellular growth phases
Adhesion speeds (h-1) (regression coefficient)
Static
Dynamic
Lag
0.8704 (0.2669)
0.9302 (0.2601)
Exponential
0.0736 (0.3096)
0.1805 (0.3277)
Stationary
0.0019 (0.2700)
0.0050 (0.3184)
Decline
0.1801 (0.2008)
0.7890 (0.2022)

Int. J. Res. BioSciences 38
min incubation durations under static and dynamic conditions (Figure 2). Abundances of A. hydrophila
adhered to the control substrate during the decline growth phase varied from 0 to 4 CFU/cm².
Adhered cells after the action of NaOCl were almost few (Figure 2).
Figure 2: Temporal evolution of adhered A. hydrophila under static and dynamic conditions,
and after the action of NaOCl (A, A’: Lag phase; B, B’: Exponential phase; C, C’: Stationary
phase, D, D’: Decline phase).
Abundance of A. hydrophila adhered to polyethylene after H2O2 disinfection
The densities of adhered A. hydrophila ranged from 5 to 2469 CFU/cm² after the action of H2O2. The
maximum abundance of adhered A. hydrophila was recorded during the stationary cell growth phase
under static condition, in the presence of 5 ‰ H2O2 and this after 720 min incubation duration (Figure
3). Adhered A. hydrophila has been sometimes always partially decimated by H2O2.
With cells coming from the lag growth phase, the abundance of A. hydrophila adhered to the control
substrate increase with the increasing of the incubation duration. The highest cell density was
recorded after 720 min of adhesion test under dynamic condition. After action of H2O2, the densities of
adhered A. hydrophila ranged from 5 to 120 CFU/cm². The effectiveness of H2O2 decreased with the
duration of the adhesion test. The highest cell abundance was recorded in the presence of 5 ‰ H2O2

Int. J. Res. BioSciences 39
after 720 min of adhesion test under dynamic regime. The lower density of adhered cells was
observed in the presence of 15 ‰ H2O2 after 180 min of an adhesion test under static and dynamic
conditions (Figure 3).
Abundances of A. hydrophila adhered to control substrate during the exponential growth phase
fluctuated between 288 and 909 CFU/cm². The highest cell abundance was recorded under dynamic
condition. After disinfection test, it was noted that the effectiveness of H2O2 decreased when the
duration of adhesion test increased. Abundance of adhered A. hydrophila ranged between 127 and
661 CFU/cm² (Figure 3).
With cells harvested from the stationary growth phase, abundance of A. hydrophila adhered to the
control substrate remained higher than those fragments tested for disinfection. The highest cell
density was recorded after an adhesion test under static condition. After disinfection test, abundances
of adhered A. hydrophila ranged between 50 and 2469 CFU/cm². As the duration of adhesion test
increased, it was noted that the effectiveness of H2O2 decreased. The highest density of A. hydrophila
adhered to the polyethylene was recorded in the presence of 5 ‰ H2O2 after 720 min of adhesion test
under static condition. The lowest was observed after 180 min of adhesion test under static condition
in the presence of 15 ‰ H2O2 (Figure 3).
Abundances of A. hydrophila adhered to the control substrate during the decline cell growth phase
varied from 43 to 1669 CFU/cm². The maximum abundance was recorded under dynamic condition
after 720 min of adhesion test. Abundances of adherent cells ranged between 20 and 1474 CFU/cm²
after disinfection test. The maximum abundance of A. hydrophila adhered to the polyethylene was
recorded in the presence of 5 ‰ H2O2 after 720 min whereas the lowest was registered in the
presence of 15 ‰ H2O2 after 180 min. Both abundances were noted under dynamic condition (Figure
3).
Figure 3: Temporal evolution of adhered A. hydrophila under static and dynamic conditions,
and after the action of H2O2 (A, A’: Lag phase; B, B’: Exponential phase; C, C’: Stationary
phase, D, D’: Decline phase).

Int. J. Res. BioSciences 40
Figure 4: Freundlich Isotherms for A. hydrophila under static condition in the presence of
NaOCl and H2O2 [lag (A’1, A’2), exponential (B’1, B’2), stationary (C’1, C’2), and decline
(D’1, D’2)]

Int. J. Res. BioSciences 41
Figure 5: Freundlich Isotherms for A. hydrophila under dynamic condition in the presence of
NaOCl and H2O2 [lag (A1, A2), exponential (B1, B2), stationary (C1, C2), and decline (D1, D2)]

Int. J. Res. BioSciences 42
Freundlich isotherms of A. hydrophila
Freundlich isotherms were constructed by considering the number of A. hydrophila adhered to the
substrate and subjected to the test of disinfection, and that obtained without exposure to disinfectants
for each stage of cell growth and each experimental condition. They are shown in figures 4 and 5. It
can be noted that whether A. hydrophila is in a static or dynamic incubation condition or coming from
a definite cell growth stage, the appearance of the isotherms differ from one disinfectant to another.
The linearity coefficient l/n which is related to the adsorption intensity ranged from 0.001 to 31.423
and from 0.002 to 16.286 for NaOCl, from 0.786 to 1544.335 and from 0.024 to 533.932 for H2O2
respectively under dynamic and static incubation conditions. For the whole disinfectant concentrations
of NaOCl used, the adsorption coefficient Kf which is related to the adsorption capacity ranged
between 1 and 403, and between 1 and 1627 adhered A. hydrophila respectively under static and
dynamic incubation conditions; For the whole disinfectant concentrations of H2O2 used, it ranged
between 0 and 148, and between 0 and 813 adhered A. hydrophila respectively (Table 2).
The lowest adsorption coefficient after NaOCl treatment was obtained with cell harvested from the
decline and stationary cell growth phases. The same observations were made with H2O2 (Table 2).
When considering each experimental condition or cells harvested from each growth stage, the
adsorption coefficient of A. hydrophila adhered to polyethylene was relatively higher after NaOCl
treatment than that after H2O2 (Table 2). It was also noted that for the whole cell growth phases and
the whole disinfectant concentrations used, the adsorption coefficient values were relatively higher
under static than dynamic incubation condition (Table 2).
Correlation between the abundances of adhered A. hydrophila and incubation durations or
disinfectant concentrations
Spearman "r" correlation coefficients between the abundances of adhered A. hydrophila and
incubation durations for each concentration disinfectant and under each experimental condition were
assessed and are presented in (Table 3). It is noted that the increase of the incubation durations
caused a significant (P<0.01) decrease of the efficiency of each of the disinfectant concentration. This
could result in higher abundances of adhered A. hydrophila as the duration of the cell adhesion
process increased.
Spearman "r" correlation coefficients between abundances of adhered A. hydrophila and disinfectant
concentrations for each incubation duration and under each experimental condition were also
assessed. Under static as well as dynamic condition, it was noted that the effectiveness of NaOCl and
H2O2 on A. hydrophila adhered to polyethylene increased, which leads to a significant decrease
(P<0.01) in the abundance of bacteria adhered after disinfection treatment.
The degrees of relationship between the disinfectant concentrations and abundances adhered A.
hydrophila harvested from each growth stage were also assessed. It resulted that an increase of the
disinfectant concentration significantly increased (P<0.01) the abundance of A. hydrophila adhered to
the substrate, with cell harvested from each growth phase.
Comparison amongst abundances of adhered A. hydrophila harvested from different cell
growth stages
The H test of Kruskal-Wallis was performed in order to compare the abundances of adhered A.
hydrophila harvested from different cell growth stages and considering each disinfectant
concentration. It showed that there was an overall significant difference (P<0.05) between the mean
abundances of A. hydrophila adhered to polyethylene for each disinfectant concentrations at different
growth stages. The pair two by two comparisons of the mean abundances were then performed using
the U test of Mann-Whitney. It was noted that at each cell growth stage, there was a significant
difference (P<0.05) between the mean abundances of adhered A. hydrophila after action of various
concentrations of NaOCl with cells coming from each the growth phase. With different concentrations
of H2O2, a significant difference (P<0.05) was observed only with cells harvested from lag growth
phase (Table 4).
The growth curve of A. hydrophila obtained shows a lag growth phase from 0 to 2 hours, an
exponential growth phase from 2 to 13 hours, a stationary growth phase from 13 to 22 hours, and a

Int. J. Res. BioSciences 43
decline growth phase begins from the 22th hour. It described a hyperbola of four phases. Bacterial
growth is an orderly increment of all the components of the bacterium [31]. It leads to an increase in the
number of bacteria. During growth, there is, on one hand, a depletion of nutrients in the culture
medium and, on the other hand, an enrichment of products of metabolism, that are toxic. During the
lag growth phase, the growth rate is nil. Bacteria adapt and synthesize the enzymes necessary to
metabolize new substrates. The exponential growth phase corresponds to the period of nutrient
utilization and duplication of cell number. The stationary growth phase is the period when the growth
rate becomes nil. In fact, the bacteria multiply compensating those who die. The decline growth phase
is the time when all food resources are exhausted. There is accumulation of toxic metabolites. There
is a decrease of viable organisms and an occurring of cell lysis by the action of endogenous
proteolytic enzymes. However, there is a persistant growth leading to the release of substances
during lysis (cryptic growth) [31].
The adhesion of microorganisms to surfaces is the first step in biofilm formation which is a form of
microbial life in aquatic environments [32]. The latter is a source of biocontamination problems in
various domains such as health, environment, food industry, water purification [30, 33, 34]. Adhesion is
governed by physico-chemical interactions of van der Waals type and Lewis’s acid-base. Fluctuating
of speeds of adhesion of A. hydrophila observed during different stages of growth in static and
dynamic conditions could be explained by changes in the physiology of bacteria in each growth stage
[35, 36].
This study showed that whatever the experimental condition, the abundances of adhered A.
hydrophila at all stages of growth are generally lower in the presence of NaOCl and H2O2 than in the
absence of these disinfectants. Unlike antibiotics, the mode of action of disinfectants is characterized
by a lack of specificity, and remains partially understood. Three possible action phases are; the
binding to the bacterial wall, conditioned by a disinfectant concentration and the Brownian movement
of bacteria [37]. The phenomenon is chemical or electrical. The penetration through the wall and the
membrane is conditioned by solubility and ionization of steric hindrance. The action itself, can affect
different targets including the cytoplasmic membrane and organelles [25]. In addition, the standard
AFNOR T 72.101 defines disinfection as an operation that permits the elimination or killing of micro-
organisms and/or inactivates undesirable viruses carried by contaminated inert environments, based
on the already fixed objectives on a momentary resultant [38]. The result of this operation is limited to
microorganisms present at the time of the operation. Considering separately, each experimental
condition, it was noted on one hand that the increase in incubation durations lead to a very significant
decrease (P<0.01) in the effectiveness of each disinfectant concentrations. This leads to a rise in the
abundances of adhered A. hydrophila. In fact, a biofilm can develop within hours, allowing bacteria
therein to become resistant to external agents causing any eventual contamination [39, 40]. On the other
hand, for each incubation duration, the action of NaOCl and H2O2 on A. hydrophila adhered to
polyethylene increased significantly (P<0.01). The action of these disinfectants is explained by the
fact that they are chemically more reactive molecules on biofilms [41]. Furthermore, this variation of the
reaction of A. hydrophila with disinfectants may be related to changes in their surface due to a change
in their growth stage [20]. Whether in the presence of NaOCl or H2O2, a significant difference (P<0.05)
between the mean densities of adhered A. hydrophila to different stages of growth was recorded. This
difference could be due to a nutritional insufficiency that suffered A. hydrophila and that could cause a
change in the growth rate [42, 43, 44]. Same at each growth stage there was a significant difference
(P<0.05) between the mean abundances of A. hydrophila adhered after action of various
concentrations of NaOCl. It is important to remember that the bacteria in a biofilm have characteristics
very different from their planktonic counterparts including the production of exopolymers [45], a
significant increase in their resistance to antimicrobial agents and environmental stress [46, 47]. The
exopolymeric matrix that presents itself as a mechanical barrier, by reducing the rate of penetration of
compounds from the environment through the biofilm protects the bacterial cells enclosed in the
biofilm. This explains the fact that the increase in the concentration of disinfectants for each growth
stage resulted in a significant increase (P<0.01) in the abundance of adhered A. hydrophila to the
substrate.
No significant difference between the mean densities of adhered A. hydrophila under static condition
and those obtained under dynamic condition has been noted (P>0.05). In fact, the conservation of
water quality is even easier than the residence time in the distribution network is short. The average
residence time in the distribution network can be in the order of a few days, but some amounts of
water can stagnate over ten days in areas of the distribution network where the flow is low or when

Int. J. Res. BioSciences 44
water demand is almost nil (during holiday periods for example). A sudden increase in the flow rate
can cause tearing of the biofilm and therefore a transient but significant deterioration of water quality
in distribution networks [48]. It has been indicated that the stagnation or slow flow speeds favors
corrosion and biofilms deposits or conditioning film by adsorption of macromolecules which can lead
to the modification of the surface properties of the substrate, and therefore, promote bioadhesion of
the pipe surfaces. These deposits appear as soon as the water speed is less than 0.01 ms-1 and
disappear beyond 0.1 ms-1[49]. However, for a given biofilm, a turbulent flow is also beneficial in
promoting the transport of nutrients and micro-organisms [50]. This allows the renewal of the
environment and improves the stability of biofilms [51].
Whether each experimental condition or each growth stage is considered, the adsorption coefficient Kf
of A. hydrophila adhered to polyethylene is relatively higher in the presence of NaOCl than in the
presence of H2O2. This could be explained by the fact that NaOCl is made up of more chemically
reactive molecules on biofilms than H2O2 [41]. In addition, considering A. hydrophila at a growth stage
in the presence of a given disinfectant, the adsorption coefficient Kf is relatively higher in static than
dynamic condition. A. hydrophila adhered to polyethylene in dynamic condition is more sensitive to
both disinfectants than that adhered under static condition. This could be explained by the structure of
the adhered bacteria which depend on hydrodynamic regime [52].
Conclusion
Abundances of A. hydrophila adhered at all cell growth stages is relatively low in the presence of
NaOCl and H2O2 than in the absence of these disinfectants, whatever the experimental condition. In
the presence of NaOCl as well as in the presence of H2O2, there is a significant difference between
the mean densities of A. hydrophila adhered to different growth stage. The effectiveness of each
concentration of NaOCl and H2O2 on A. hydrophila adhered to polyethylene decreased as the
duration of the adhesion test increased. Although the adsorption coefficient obtained from Freundlich
isotherms was relatively higher in static than in dynamic condition, no significant difference was
observed between the mean abundances of adhered A. hydrophila under these two experimental
conditions. Overall, NaOCl seems more effective on A. hydrophila adhered to the polyethylene than
H2O2. Furthermore, A. hydrophila adhered to the polyethylene in dynamic condition seems more
sensitive to the two disinfectants than that adhered in static condition.
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Int. J. Res. BioSciences 48
Table 2: Values of adsorption coefficient Kf (adhered A. hydrophila /ml of disinfectant) and linearity coefficient l/n of isotherms under static and
dynamic conditions.
Disinfectant and
its
concentrations
Experimental condition, cell growth phase and Freundlich coefficient
Static
Dynamic
Adsorption (Kf)
Linearity (ln)
Adsorption (Kf)
Linearity (ln)
Lag
Expo.
Stat.
Decl.
Lag
Expo.
Stat.
Decl.
Lag
Expo.
Stat.
Decl.
Lag
Expo.
Stat.
Decl.
NaOCl
0.5‰
2
403
3
1
31.42
0.27
0.40
0.06
3
1627
3
1
16.29
1.26
0.34
0.02
1‰
1
148
3
1
11.29
1.68
0.18
0.02
298
55
3
1
2.51
0.45
0.19
0.02
1.5‰
403
55
1
1
2.29
1.12
0.07
0.00
3
20
1
1
0.26
1.18
0.04
0.00
H2O2
5‰
2
8
3
0
1.43
4.21
3.14
1544.33
2
2
1
0
35.44
6.8
4.86
533.93
10‰
3
1
5
0
12.09
3.80
6.31
1306.37
5
1
4
0
31.40
22.12
1.05
397.67
15‰
148
4
6
0
27.66
2.93
0.79
369.44
813
2
4
0
14.78
7.69
0.02
168.57
Table 3: Spearman "r" correlation coefficients between the abundances of adhered A. hydrophila and incubation durations for each concentration
of disinfectant and each experimental condition.
Experimental condition
Disinfectant concentrations
NaOCl
H2O2
0.5 ‰
1 ‰
1.5 ‰
5 ‰
10 ‰
15 ‰
Static
0.273
-0.405**
-0.533**
0.003
-0.079
-0.021
Dynamic
-0.542**
-0.700**
-0.710**
0.066
0.391*
-0.568**
** P <0.01 ddl=15
Table 4: Comparison amongst abundances of A. hydrophila harvested from different cell growth stages in the presence of each disinfectant
Cell growth phases
A. hydrophila
NaOCl
H2O2
Lag
P=0.007*
P=0.034*
Exponential
P=0.000*
P=0.114
Stationnary
P=0.000*
P=0.185
Decline
P=0.015*
P=0.963
* P<0.05 ddl=92