Membrane Damage and Microbial Inactivation by Chlorine in the Absence and Presence of a Chlorine-Demanding Substrate

Abstract

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium
drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal
injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed
a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization
of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore,
there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter
to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative
bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in
the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell
inactivation by modulating the access of chlorine to the key targets within the cell.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2005, p. 5022–5028 Vol. 71, No. 9
0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.9.5022–5028.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Membrane Damage and Microbial Inactivation by Chlorine in the
Absence and Presence of a Chlorine-Demanding Substrate
R. Virto, P. Man˜as, I. A
´lvarez, S. Condon, and J. Raso*
Tecnologı´a de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, 50.013 Zaragoza, Spain
Received 4 November 2004/Accepted 29 March 2005
The relationship between cell inactivation and membrane damage was studied in two gram-positive organ-
isms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and
Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy
broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small
amounts of organic matter to the chlorination medium drastically increased the resistance of both types of
microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves
for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not
detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a
relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive
permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those
needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane perme-
abilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic
membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Ex-
posure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine
and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that
bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets
within the cell.
Chlorination is one of the most widely used processes for
microbial control (5) in both drinking water and wastewater
processing (16). Chlorine is a powerful antimicrobial substance
due to its potential oxidizing capacity. In addition to drinking
water disinfection, there are a number of other commercial
uses for chlorine in the food industry, including reduction of
microbial populations on the surfaces of raw foods, such as
fruits and vegetables, and sanitation of surfaces in food pro-
cessing environments (5, 7). In general, disinfectants such as
chlorine are used at very high concentrations in order to attain
a rapid rate of killing. At the concentrations used, it is difficult
for microorganisms to survive. However, the use of such high
concentrations increases the risk of formation of potentially
hazardous by-products or the production of off-tastes and
odors, which are the main drawbacks of chlorination (13, 17).
Conversely, at low chlorine levels, microorganisms that survive
the treatment may be injured rather than inactivated (8). Un-
der suitable conditions injured cells might repair cellular dam-
age and recover (10). An appreciation of the nature of suble-
thal injury and repair is therefore important in devising
chlorination strategies and in developing combination treat-
ments with synergistic actions against the target microorgan-
isms. The mechanisms of action of chlorine on microorganisms
have been widely investigated (1, 3, 19). Nevertheless, the
mechanism by which chlorine exerts its lethal effect has never
been fully elucidated. Moreover, the occurrence of sublethal
injury caused by chlorine treatments is scarcely known.
Effective microbial control by chlorine requires appropriate
disinfection design criteria to ensure protection of public
health and to minimize unwanted effects of the chlorination
process. A better understanding of the way that chlorine kills
cells would help in defining effective chlorine treatments and in
optimizing strategies for chlorination.
The purpose of this study was to investigate the occurrence
of sublethal injury and the relationship between membrane
damage and loss of viability in two gram-positive and two
gram-negative microorganisms after exposure to chlorine in
the absence and presence of a chlorine-demanding substrate.
MATERIALS AND METHODS
Bacterial strains and growth conditions. Escherichia coli STCC 471, Yersinia
enterocolitica STCC 4315, Listeria monocytogenes ATCC 15313, and Bacillus
subtilis NCTC 10073 were used in this study. E. coli,Y. enterocolitica, and L.
monocytogenes were grown in tryptic soy broth with 0.6% yeast extract (TSBYE)
(Biolife, Milan Italy), and B. subtilis was grown in nutrient broth (Biolife, Milan
Italy). A subculture of each strain was obtained by inoculating a test tube
containing 5 ml of sterile broth with a single colony and incubating it overnight
at 37°C. A flask containing 50 ml of broth was inoculated with the subculture to
obtain a concentration of 10
6
CFU/ml. The bacteria were grown to the stationary
phase with agitation at 37°C. Before treatment, microorganisms were centrifuged
at 6,000 ⫻gfor 5 min at 4°C. The broth was removed, and the cell pellet was
rinsed and resuspended in a phosphate buffer solution (0.01 M, pH 7). This
washing procedure was repeated several times until the supernatant did not
demand chlorine. The chlorine demand was estimated by the N,N-dimethyl-p-
phenylenediamine colorimetric method (4). As small variations in the cell con-
centration could affect chlorine depletion and therefore chlorine efficacy, the
microbial concentration was determined by measuring absorbance at 620 nm.
Microbial suspensions were diluted in distilled water or in tryptic soy broth
(TSB) (1,500 ppm, corresponding to 1,120 ppm of organic load calculated from
* Corresponding author. Mailing address: Tecnologı´a de los Ali-
mentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel
Servet 177, 50.013 Zaragoza, Spain. Phone: 34 976 76 15 81. Fax: 34
976 76 15 90. E-mail: jraso@unizar.es.
5022

the formulation) to obtain a concentration of 6 ⫻10
8
⫾1⫻10
8
CFU/ml for
inactivation experiments. Before each experiment absorbance measurements
were confirmed by plate counting. All experiments were performed in demand-
free glassware.
Stock chlorine solutions. Free chlorine solutions were prepared from reagent
grade chemicals (10% sodium hypochlorite from Panreac, Barcelona, Spain). A
stock solution (100 ppm available chlorine) was prepared using deionized dis-
tilled water that did not demand chlorine. Deionized distilled water was also used
to prepare all other hypochlorite solutions. The initial chlorine concentrations
ranged from 0.3 to 30 ppm. The initial free chlorine (hypochlorous acid plus
hypochlorite ion) concentration was determined by the N,N-dimethyl-p-phenyl-
enediamine method (4).
Chlorine decay assays. Once microorganisms were added to the chlorine
solutions, samples were taken after different contact times. Free chlorine con-
centrations were determined by the method described above. All determinations
were done in duplicate, and the results were expressed as the average values.
Microbial inactivation experiments. Microbial resistance to chlorine was eval-
uated by adding 0.1 ml of a microbial suspension in distilled water or in 1,500
ppm TSB to 1.5-ml conical flasks containing 0.9 ml of a corresponding chlorine
solution at 20°C. Samples were collected after different treatment times ranging
from 0.08 to 30 min. After the desired contact time, appropriate serial dilutions
were prepared in TSBYE. It was previously determined that TSBYE neutralized
the residual chlorine in the corresponding dilutions.
EDTA treatment. Samples (0.1 ml) of a suspension of E. coli were added to 0.9
ml of distilled water that contained 20 mM of EDTA disodium salt. After 30 min
of incubation the EDTA was removed by centrifugation, and the EDTA-treated
microorganisms were resuspended in distilled water or in TSB (150 ppm). The
chlorine resistance of these EDTA-treated microorganisms was determined as
indicated above.
Counting of survivors. The numbers of survivors of E. coli,Y. enterocolitica,
and L. monocytogenes were determined in tryptic soy agar with yeast extract
(TSAYE) or in TSAYE with 4, 2, and 5% NaCl, respectively, and the number of
survivors of B. subtilis was determined in nutrient agar (NA) or in NA with 6%
NaCl. The concentrations used to detect sublethal injury correspond to the
highest NaCl concentrations that did not affect the growth of untreated cells.
Plates were incubated at 37°C for 24 to 72 h, and after incubation, colonies were
counted with an improved image analyzer automatic counter (Protos, Analytical
Measuring Systems, Cambridge, United Kingdom) as described elsewhere (2).
The number of sublethally injured bacteria corresponded to the difference be-
tween the counts obtained in the nonselective medium (TSAYE or NA) and the
counts obtained in the selective medium (medium supplemented with NaCl).
Leakage of UV-absorbing substances. To quantify the intracellular material
released from the cells, untreated and treated concentrated samples (6 ⫻10
8
⫾
1⫻10
8
CFU/ml) were centrifuged at 6,000 ⫻gfor 10 min. The UV absorbance
of the supernatant was measured at 260 and 280 nm with a spectrophotometer
(UNICAM UV 500 UV-visible spectrometer).
Determination of propidium iodine uptake. The integrity of the cytoplasmic
membranes of chlorine-treated cells was determined using propidium iodide (PI)
(Sigma-Aldrich). Untreated and chlorine-treated cells were centrifuged, the su-
pernatants were removed, and the cell pellets were rinsed and resuspended in
distilled water to an optical density at 620 nm of approximately 0.4 for E. coli or
1 for L. monocytogenes, corresponding to approximately 10
8
cells per ml. PI was
added to a final concentration of 2 ␮g/ml. After incubation for 10 min, samples
were centrifuged and washed in distilled water to remove the excess dye. Fluo-
rescence was measured with a spectrofluorophotometer (model TECAN; Ge-
nius) at an excitation wavelength of 535 nm and an emission wavelength of 625
nm. Fluorescence data for each sample were normalized with the optical density
at 620 nm; the values obtained for untreated cells were subtracted from all
experimental values.
RESULTS
Microbial chlorine resistance in distilled water and TSB
(150 ppm). In order to study chlorine resistance, the initial free
chlorine concentrations were varied depending on the intrinsic
resistance of the microorganism tested. In distilled water, the
chlorine sensitivities of the gram-negative microorganisms E.
coli and Y. enterocolitica were investigated by using concentra-
tions of 0.3 to 0.7 ppm and 0.8 to 1 ppm, respectively, while the
chlorine sensitivities of the gram-positive microorganisms B.
subtilis and L. monocytogenes were investigated by using con-
centrations of 0.8 to 1.2 ppm and 0.6 to 0.9 ppm, respectively.
When cells were exposed to the ranges of chlorine concentra-
tions indicated above for 2 min, the gram-positive microorgan-
isms showed greater resistance (Fig. 1A). E. coli was more
sensitive than Y. enterocolitica, and L. monocytogenes was more
sensitive than B. subtilis.
The presence of organic matter in the treatment medium
drastically increased the chlorine resistance of the four micro-
organisms investigated. In the presence of organic matter the
free chlorine concentrations assayed ranged from 10 to 30
ppm. The increase in resistance was especially marked for the
gram-negative microorganisms, which were even more chlo-
rine resistant than the gram-positive microorganisms (Fig. 1B).
After contact for 2 min, cell death began at initial chlorine
concentrations of more than 20 and 10 ppm for gram-negative
and gram-positive microorganisms, respectively. As observed
FIG. 1. Microbial inactivation by treatment with different initial
chlorine concentrations for 2 min in the absence of TSB (A) and in the
presence of TSB (150 ppm) (B) at 20°C. Symbols: F,B. subtilis;■,L.
monocytogenes;E,Y. enterocolitica;䊐,E. coli.
VOL. 71, 2005 MEMBRANE DAMAGE AND CHLORINE INACTIVATION 5023

in distilled water, Y. enterocolitica was more resistant than E.
coli; however, B. subtilis was more resistant than L. monocyto-
genes.
Inactivation kinetics and occurrence of sublethal injury in
distilled water and TSB (150 ppm). Examples of survival
curves showing the inactivation of the four microorganisms in
distilled water are shown in Fig. 2A and B. In order to show
whether chlorine caused sublethal injury, survival curves for
the treated microorganisms recovered in a selective medium
are also included. As shown in Fig. 2, the survival curves for
gram-negative microorganisms showed that initially there was
very fast linear inactivation, which was followed by zero-slope
tailing (Fig. 2A), whereas the survival curves for gram-positive
microorganisms were concave upward (Fig. 2B).
For both gram-negative bacteria there was no difference
between the counts in nonselective plating medium and the
counts in selective plating medium (Fig. 2A). However, it was
found that chlorine caused sublethal injury in gram-positive
microorganisms since a greater reduction in viability was ob-
served when cells were recovered in the selective medium.
After 1 min of treatment more than 99% of the B. subtilis and
L. monocytogenes survivors were injured and therefore unable
to grow in the presence of sodium chloride. Similar results
were observed for various initial chlorine concentrations (data
not shown).
In all cases investigated the levels of chlorine consumption
for the populations of the four microorganisms were found to
be similar. Microbial suspensions in distilled water exerted a
high chlorine demand, and the free chlorine was almost com-
pletely consumed by the bacterial cell suspensions during the
first moments of the treatment (data not shown).
Examples of survival curves in TSB (150 ppm) correspond-
ing to inactivation of the four microorganisms investigated
recovered in nonselective and selective plating media are
shown in Fig. 3. The presence of organic matter in the treat-
ment medium modified the shape of the survival curves for the
gram-negative microorganisms, which had a shoulder followed
by a linear inactivation. Shoulders in survival curves have been
attributed to accumulation of repairable damage that becomes
irreversible only beyond a critical level. However, for both
gram-negative and gram-positive microorganisms there were
no differences between the counts in nonselective medium and
the counts in selective plating medium. Therefore, in the pres-
ence of organic matter, chlorine did not injure cells before they
were completely inactivated. Similar behavior was observed in
experiments performed with different chlorine concentrations.
TSB (150 ppm) in solutions containing bacterial suspensions
was found to exert a rapid chlorine demand in the first 1 min
of treatment, followed by a slow demand with a longer contact
time. For the four microorganisms, the levels of residual free
chlorine during the treatment were always higher than 3 ppm.
Leakage of UV-absorbing substances caused by chlorine
treatment. The values for absorbance at 260 nm for superna-
tants of E. coli and L. monocytogenes suspensions treated in
distilled water and TSB (150 ppm) are shown in Fig. 4A and B.
The results obtained at 280 nm were similar (data not shown).
Increasing the chlorine concentration resulted in increased
levels of UV-absorbing substances for both microorganisms
when they were treated in distilled water. However, leakage of
UV-absorbing substances began to be remarkable at chlorine
concentrations higher than the concentration necessary to in-
activate more than 99.9% of the populations of both microor-
ganisms.
When TSB was added to the treatment medium, no leakage
of UV-absorbing substances occurred even at chlorine concen-
trations as high as 100 ppm for the two microorganisms tested.
Uptake of PI after chlorine treatment. Propidium iodide is
commonly used as an indicator of cytoplasmic membrane dam-
age. PI is able to enter cells only if the membrane is perme-
abilized. Once inside the cytoplasm, it binds to single- and
double-stranded nucleic acids, yielding fluorescence in the red
wavelength region. Data for the uptake of PI by chlorine-
FIG. 2. Survival curves corresponding to microbial inactivation by
chlorine in distilled water at 20°C. (A) Symbols: F,Y. enterocolitica, 0.7
ppm chlorine, enumeration in TSAYE; E,Y. enterocolitica, 0.7 ppm
chlorine, enumeration in TSAYE containing 2% NaCl; ■,E. coli, 0.5
ppm chlorine, enumeration in TSAYE; 䊐,E. coli, 0.5 ppm chlorine,
enumeration in TSAYE containing 4% NaCl. (B) Symbols: F,L.
monocytogenes, 0.7 ppm chlorine, enumeration in TSAYE; E,L.
monocytogenes, 0.7 ppm chlorine, enumeration in TSAYE containing
5% NaCl; ■,B. subtilis, 1 ppm chlorine, enumeration in NA; 䊐,B.
subtilis, 1 ppm chlorine, enumeration in NA containing 6% NaCl.
5024 VIRTO ET AL. APPL.ENVIRON.MICROBIOL.

treated cells of E. coli and L. monocytogenes when they were
treated in distilled water and TSB (150 ppm) are shown in Fig.
5A and B, respectively. The uptake of propidium iodide exhib-
ited the same pattern as the leakage of UV-absorbing material
shown in Fig. 4A and B. When treated in distilled water, both
microorganisms took up the dye at chlorine concentrations
above 1 ppm. Since chlorine concentrations below 1 ppm for 2
min inactivated more than 99.9% of the cells of both micro-
organisms, the onset of cell staining occurred once the cells
were dead. No uptake was observed in the two microorganisms
when they were treated in TSB (150 ppm), even at high chlo-
rine concentrations.
Chlorine resistance of EDTA-treated E. coli cells. It is well
known that the outer membrane of gram-negative bacteria has
a clear role in modulating the accessibility of low-molecular-
weight substances to the cytoplasm. We studied whether pre-
vious treatment of E. coli cells with an outer membrane-per-
meabilizing agent, such as EDTA, could sensitize cells against
chlorine. Figure 6 shows that preincubation with EDTA dras-
tically decreased the chlorine resistance of E. coli in both
distilled water and TSB (150 ppm). Sublethal injury was not
detected in any case (data not shown).
DISCUSSION
Results obtained in this investigation showed that there were
large differences in chlorine resistance depending on the mi-
croorganism investigated and the composition of the treatment
FIG. 3. Survival curves corresponding to microbial inactivation by
chlorine in the presence of TSB (150 ppm) at 20°C. (A) Symbols: F,Y.
enterocolitica, 20 ppm chlorine, enumeration in TSAYE; E,Y. entero-
colitica, 20 ppm chlorine, enumeration in TSAYE containing 2%
NaCl; ■,E. coli, 20 ppm chlorine, enumeration in TSAYE; 䊐,E. coli,
20 ppm chlorine, enumeration in TSAYE containing 4% NaCl.
(B) Symbols: F,L. monocytogenes, 15 ppm chlorine, enumeration in
TSAYE; E,L. monocytogenes, 15 ppm chlorine, enumeration in
TSAYE containing 5% NaCl; ■,B. subtilis, 150 ppm chlorine, enu-
meration in NA; 䊐,B. subtilis, 150 ppm chlorine, enumeration in NA
containing 6% NaCl.
FIG. 4. Leakage of UV-absorbing substances from E. coli and L.
monocytogenes treated with chlorine for 2 min. The graphs show rep-
resentative results. (A) Symbols: ■,E. coli treated in distilled water; F,
E. coli treated in TSB (150 ppm). (B) Symbols: ■,L. monocytogenes
treated in distilled water; F,L. monocytogenes treated in TSB (150
ppm).
VOL. 71, 2005 MEMBRANE DAMAGE AND CHLORINE INACTIVATION 5025

medium. In distilled water gram-negative microorganisms (E.
coli and Y. enterocolitica) were more chlorine sensitive than
gram-positive microorganisms (B. subtilis and L. monocyto-
genes). The microbial resistance drastically increased when
TSB was present in the treatment medium. This effect was
more marked for the two gram-negative microorganisms,
which exhibited different inactivation kinetics and became
even more chlorine resistant than the two gram-positive mi-
croorganisms.
The protective effect of organic matter against chlorine has
been described previously (6). This effect has been attributed
to the higher chlorine demand of organic compounds, which
results in a rapid decline in the available free chlorine (5, 9,
15). However, in experiments conducted in this investigation
the residual chlorine concentration in the presence of organic
matter was much higher than the chlorine concentration nec-
essary to completely inactivate the microbial population in
distilled water. Therefore, the protective effect cannot be at-
tributed solely to the chlorine demand of TSB but may be due
to effective stabilization of some cellular structures.
Another aspect observed in this study was the different in-
activation kinetics depending on the microorganism and the
treatment medium. In distilled water most inactivation of
gram-negative microorganisms occurred during the first 1 min
of treatment, and the counts remained unchanged when the
treatment was extended. However, in TSB (150 ppm) the sur-
vival curves had a shoulder, followed by linear inactivation.
The occurrence of a shoulder in survival curves with different
lethal agents has been attributed to accumulation of damage
that becomes irreparable only beyond a critical level. The oc-
currence of sublethal injury to the membrane was studied by
the selective plating technique using sodium chloride as the
inhibitory compound. In this investigation it was found that
chlorine did not cause sublethal injury in gram-negative micro-
organisms when they were suspended both in distilled water
FIG. 5. Permeability of chlorine-treated cells for 2 min to pro-
pidium iodine. The graphs show representative results. (A) Symbols:
■,E. coli treated in distilled water; F,E. coli treated in TSB (150
ppm). (B) Symbols: ■,L. monocytogenes treated in distilled water; F,
L. monocytogenes treated in TSB (150 ppm). OD, optical density; Ex,
excitation; Em, emission. FIG. 6. Influence of preincubation with EDTA (20 mM) for 30 min
on E. coli resistance to chlorine. (A) Solid line, native cells treated in
distilled water, 0.4 ppm chlorine; dashed line, cells preincubated with
EDTA and treated in distilled water, 0.4 ppm chlorine. (B) Solid line,
native cells treated in TSB (150 ppm), 15 ppm chlorine; dashed line,
cells preincubated with EDTA and treated in TSB (150 ppm), 15 ppm
chlorine.
5026 VIRTO ET AL. APPL.ENVIRON.MICROBIOL.

and in TSB (150 ppm). Therefore, we concluded that the
occurrence of a shoulder in inactivation curves for gram-neg-
ative bacteria in the presence of organic matter does not seem
to be due to cellular injury.
Chlorine is generally considered to be a nonselective oxidant
which reacts avidly with a variety of cellular components and
affects metabolic processes (14). The cytoplasmic membrane
has been proposed to be a possible key target involved in
bacterial inactivation by chlorine (19), since alterations in its
permeability after chlorination have frequently been described.
Sensitivity to salt in the recovery medium is recognized as a
symptom of damage to the cytoplasmic membrane (10, 19). In
this investigation no sublethally injured cells were detected in
most cases, and only gram-positive cells treated in distilled
water showed a significant degree of sublethal injury. There-
fore, only in this case did sublethal damage to the membrane
precede cell death. In other words, cells were able to repair
their membrane when they were recovered in an adequate
plating medium (TSAYE). In contrast, for gram-negative cells
and for cells treated in the presence of organic matter, damage
to the membrane was either irreparable or nonexistent.
To further study the nature of the membrane damage, the
leakage of intracellular substances in the gram-positive organ-
ism L. monocytogenes and the gram-negative organism E. coli
was investigated. We observed extensive leakage of UV-ab-
sorbing material after chlorine treatment in water. However,
addition of small amounts of organic matter to the treatment
medium completely prevented the leakage of UV-absorbing
material, suggesting that there was protection of the cell walls
for both the gram-positive and gram-negative microorganisms
investigated.
Since the molecular size of RNA and proteins or peptides,
which are the main molecules detected by the UV absorbance
measurements, is quite large, experiments with propidium io-
dide were also carried out. Propidium iodide is a low-molecu-
lar-mass dye (668.4 Da) which emits strong red fluorescence
when it binds to single- and double-stranded nucleic acids. Its
entrance into the bacterial cell is prevented by an intact cyto-
plasmic membrane. The results of experiments performed with
propidium iodide had the same profile as the results described
above for the leakage of UV-absorbing material, confirming
that there was permeabilization of the cytoplasmic membrane
when cells were treated in distilled water. However, membrane
permeabilization was observed when bacteria were exposed to
chlorine concentrations as high as 50 ppm, which is severalfold
higher than the concentration required for cell killing. This
indicates that extensive cytoplasmic membrane damage is not
a key event leading to cell death due to chlorine.
However, we cannot disregard the possibility that the cell
wall plays a role in the inactivation of cells by chlorine since the
presence of organic matter in the treatment medium protected
cell membranes against permeabilization and simultaneously
increased the free chlorine concentration needed to attain cell
killing. For bactericides to be effective, they must be able to
penetrate the cell envelope and attain a concentration at the
target site high enough to exert their antimicrobial action.
Results obtained in this investigation suggest that the presence
of organic matter could stabilize the envelopes and thus slow
the penetration of chlorine into the cell. Whereas the enve-
lopes of gram-positive bacteria consist of the cytoplasmic
membrane surrounded by a thick peptidoglycan wall, the en-
velopes of gram-negative bacteria posses an external layer, the
outer membrane, which provides an extra barrier against an-
timicrobial compounds. The fact that the protective effect of
organic matter was greater for gram-negative microorganisms
than for gram-positive microorganisms also suggested that
there was stabilization of the outer membrane. This hypothesis
was also supported by the occurrence of shoulders in survival
curves for gram-negative bacteria treated in the presence of
organic matter. Stabilization of the outer membrane by small
amounts of organic matter has been reported previously for
Salmonella (11). To explore the role of the outer membrane in
gram-negative bacteria, E. coli was preincubated with the outer
membrane-permeabilizing agent EDTA. EDTA provokes de-
stabilization of the outer membrane through chelation of di-
valent cations, such as calcium and magnesium (18). Divalent
cations play an important role in stabilizing the external leaflet
of the outer membrane by neutralizing the electrostatic repul-
sion between neighboring lipopolysaccharide molecules. If the
divalent cations are removed from their positions in the outer
membrane, the stability of the lipopolysaccharide layer is dis-
turbed and the permeability to several compounds is increased
(12). Preincubation with EDTA drastically increased E. coli
sensitivity to chlorine in both distilled water and TSB. Further-
more, this preincubation treatment also resulted in the disap-
pearance of the shoulders in survival curves in TSB (150 ppm),
confirming that the outer membrane is implicated in the pro-
tective effect of organic matter in E. coli.
Results obtained in this investigation support that extensive
membrane damage is not a key event in the inactivation of
bacteria by chlorine and confirm the observations of other
authors that suggest that more subtle events, such as uncou-
pling of the electron chain or enzyme inactivation either in the
membrane or in the cell interior, are involved in the bacteri-
cidal mechanism of chlorine (1, 19). However, the envelopes
play an important role in the bacterial resistance to chlorine in
the presence of organic matter, which is probably related to the
accessibility of chlorine to targets within the cell. Further in-
vestigations are required to fully elucidate these aspects and to
better exploit chlorination technology.
ACKNOWLEDGMENT
R.V. gratefully acknowledges the financial support for her doctoral
studies from the Departamento de Educacio´n y Ciencia del Gobierno
de Navarra.
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