Cells inactivated by the UV1 dose lost culturability but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis.. Wi
Trang 1Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead? Andrea Villarino1,2, Marie-Noe¨lle Rager3, Patrick A D Grimont1,2and Odile M M Bouvet2
1 Aquabiolab and 2 Unite´ de Biodiversite´ des Bacte´ries Pathoge`nes E´mergentes, INSERM U389, Institut Pasteur, Paris, France;
3 Service de Re´sonance Magne´tique Nucle´aire UMR 7576, Ecole Nationale Supe´rieure de Chimie de Paris, France
Cells that have lost the ability to grow in culture could be
defined operationally as either alive or dead depending on
the method used to determine cell viability As a
conse-quence, the interpretation of the state of nonculturable cells
is often ambiguous Escherichia coli K12 cells inactivated by
UV-irradiation with a low (UV1) and a high (UV2) dose
were used as a model of nonculturable cells Cells inactivated
by the UV1 dose lost culturability but they were not lysed
and maintained the capacity to respond to nutrient addition
by protein synthesis and cell wall synthesis The cells also
retained both a high level of glucose transport and the
capacity for metabolizing glucose Moreover, during glucose
incorporation, UV1-treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden–Meyerhof and pentose-phosphate pathways However, nonculturable cells obtained by irradi-ation with the high UV2 dose showed several levels of metabolic imbalance and retained only residual metabolic activities Nonculturable cells obtained by irradiation with UV1 and UV2 doses were diagnosed as active and inactive (dying) cells, respectively
Keywords: NMR; radiation injury; viability; metabolism; Escherichia coli
Ultraviolet irradiation has been used in the disinfection of
drinking water, wastewater and in air disinfection [1–3]
After disinfection, microorganisms are not detectable in
standard culture media in which they have been
previ-ously found to proliferate [4] Thus, a bacterium is
currently reported as dead when it does not yield visible
growth in bacteriological media for a given time [5]
However, it has been suggested that the bacterial
populations in water, when exposed to UV disinfection,
might show a decrease in culturability, but in fact they
could still be alive and able to cause disease [6]
Moreover, in aquatic systems among the various stresses
to which bacteria are submitted, solar radiation (UV-B,
290–320 nm) seems to be the most important in causing
the loss of culturability [7] Wilber and Oliver [6] showed
that, although both UV-treated Salmonella serotype
Typhimurium and Escherichia coli lost culturability in
standard culture media upon irradiation, they retained the
capacity to respond to nutrients by cell elongation in the
direct viable count (DVC) method On the other hand,
Caro et al [8] observed that UV-treated Salmonella cells
lost the capacity of cell elongation in the DVC method
and lost culturability concomitantly with pathogenicity in
mice However, these cells were also considered to be alive because they retained respiratory activity, membrane integrity and DNA integrity In a previous study, we considered that UV-treated E coli cells that retained the same activities described by Caro et al were dead because neither growth nor cell elongation or protein synthesis were detected [9] Cells could be defined operationally as alive or dead depending on the method used to determine cell viability Moreover, each method is based on criteria that reflect different levels of cellular integrity or functionality As a consequence, the interpretation of the state of cells is often ambiguous [10,11] Problems in the interpretation of the state of cells that have lost culturability are due, not only to the absence of consensus
on the definition of bacterial death but also, to the lack
of global studies showing their metabolic potential The aim of the present study was to analyze the metabolic capacities of UV-induced nonculturable E coli cells and
to determine whether the level of UV-irradiation affects their metabolic potential and responsiveness The capacity
of cells to respond to the addition of nutrients determined
by cell elongation, protein synthesis and glucose meta-bolism was analysed, as well as the effect of different aeration conditions on the regulation of metabolic fluxes through the Embden–Meyerhof and pentose phosphate pathways
Experimental procedures
Bacterial strain and growth conditions Escherichia coli K-12S sensitive to bacteriophage lambda (strain CIP 54118) from the Collection de l’Institut Pasteur (Paris, France) was used [12] Overnight cultures of E coli K-12S were maintained long-term at )80 C in Trypto Casein Soy broth (Sanofi Diagnostics Pasteur, Marnes-la
Correspondence to O M M Bouvet, Unite´ des Pathoge`nes et
Fonctions des Cellules Epithe´liales Polarise´es, INSERM U510,
Faculte´ de Pharmacie, Universite´ Paris XI, F-92296
Chaˆtenay-Malabry, France Fax: + 33 146835844, Tel.: + 33 146835843,
E-mail: odile.bouvet@cep.u-psud.fr
Abbreviations: UV1, low ultraviolet dose; UV2, high ultraviolet dose;
DVC method, direct viable count method; qDVC method,
quantita-tive direct viable count method; LB, Luria Bertani medium;
EM, Embden–Meyerhof pathway; PP, pentose phosphate pathway.
(Received 4 March 2003, revised 25 April 2003,
accepted 6 May 2003)
Trang 2Coquette, France) supplemented with glycerol (40%, v/v).
Initially, cells were grown at 37C overnight in Luria–
Bertani (LB) broth [13] transferred to a fresh medium at a
dilution of 1 : 200 and grown to a late exponential phase
(D600¼ 0.6) in aerobic conditions They were harvested by
centrifugation at 3800 g for 15 min at 9C and washed
twice in phosphate buffer pH 7.4 (11 mMK2HPO4, 5 mM
KH2PO4, 120 mMNaCl, 0.1 mMCaCl2, 0.5 mMMgSO4)
Finally, they were diluted in the same buffer to a cell density
of about 2· 107CFUÆmL)1and used immediately for UV
irradiation
UV irradiation
To obtain nonculturable cells, a cell suspension containing
(2· 107CFUÆmL)1) was irradiated as described previously
[9] Briefly, 6.5 mL of the cell suspension was placed in a
sterile glass Petri dish (11 cm diameter) and irradiated with
a 12-W (254 nm) germicidal lamp (Bioblock Scientific,
Illkirch, France) at 25C with mild agitation The lamp, at
13 cm from the Petri dish, was switched on 1 h before
utilization and the intensity of radiation at the bottom of the
Petri dish was controlled with an ultraviolet intensity meter
(Bioblock Scientific) UV dose was calculated as the product
of exposure time and the intensity at the bottom of the Petri
dish (10 mJÆmin)1Æcm)2) Cells were irradiated by two UV
doses, UV1 dose (4 mJÆcm)2) corresponding to the first dose
sufficient to obtain at least a six-log reduction (i.e
20 CFUÆmL)1) in the initial colony count and UV2 dose
(80 mJÆcm)2) inducing about a seven-log reduction (i.e
2 CFUÆmL)1) UV-treated bacteria were handled in
dark-ness After UV irradiation, treated cells were concentrated
by centrifugation (3800 g) to a cell density of about
2· 108cellsÆmL)1and used immediately in further
experi-ments For each experiment, a nonirradiated cell suspension
at the same cell density was used as an untreated control
Culturability
Samples (2 mL) of untreated or UV-treated cell suspension
were incubated with or without 440 U of catalase
(220 UÆmL)1) (Sigma) at room temperature Aliquots
(100 lL) were taken at different times and surface plated
in triplicate on LB agar supplemented with or without
catalase Some experiments used both catalase and
super-oxide dismutase (Sigma) The enzyme solutions used were
filter-sterilized through 0.22 lm pore size membrane filter,
and 0.2 mL were aseptically spread on the surface of agar
media at a concentration of 2000 U per plate Plates were
then incubated in aerobic and anaerobic conditions at
37C for 48 h
Substrate responsiveness
Substrate responsiveness of cells was determined by the
direct viable count method (DVC) [14] in the conditions
described previously with some modifications [9] Cell
samples were diluted (1/100, v/v) in LB medium containing
nalidixic acid (40 lgÆmL)1) (Sigma) Cells that exceeded at
least twice the mean length of cells before DVC were scored
as elongated The proportion of DVC positive cells was
corrected by the proportion of elongated cells detected
before the DVC method At the same time, cells incubated in the same conditions but without nalidixic acid addition were also analysed A quantitative DVC (qDVC) method was also used [15] Elongated or nonelongated substrate-respon-sive cells were selectively lysed by spheroplast formation caused by incubation with nutrients, nalidixic acid and glycine (2% final concentration) This glycine effect leads to swollen cells with a very loose cell wall The substrate-responsive cells were then lysed easily by a single freeze-thaw treatment The number of cells responding to nutrients was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation Results were expressed as percentage of substrate-responsive cells with respect to the original colony count of untreated bacteria Cell samples of DVC and qDVC were incubated in the dark at 37C for 5 h with shaking (200 r.p.m) The cells were then fixed with 3% formalin (final concentration) to be enumerated by epi-fluorescence microscopy and analysed by flow cytometry Epifluorescence microscopy and flow cytometry For cell enumeration, samples were filtered through poly-carbonate membrane filters (pore size, 0.2 lm, 25-mm diameter) (Milipore) and washed with phosphate buffer These cells were detected by staining with propidium iodide (Sigma, St Louis, MO) at 0.5 lgÆmL)1(final concentration)
or by fluorescent in situ hybridization [16] with probe EUB 338 labelled with fluorescein isothiocyanate [17] Filters were washed and mounted with Vectashield mount-ing medium (Vector, Burlmount-ingame, CA, USA) on glass microscope slides and stored in the dark at 4C until counted Cells were counted with an Olympus BX-60 epifluorescence microscope (100-W mercury lamp) with a
· 100 oil immersion fluorescent objective Cells in 24 microscopic fields per filter were enumerated and averaged (about 400 cells for nonirradiated cells) For each sample, three filters were examined and maximal deviations from the mean were calculated
Modifications of E coli size and granularity of untreated and UV-treated cells, before and after the DVC method described above, were analysed by flow cytometry [18] Duplicate samples were analysed with a Becton Dickinson model FACScan cytometer equipped with a 15-mW, air-cooled argon ion laser (488 nm) by usingCELL QUEST3.3 software The forward angle light scatter and side angle light scatter amplifier gains were set to linear and logarithmic mode, respectively For each cell sample run, data for
10 000 events were collected
Protein synthesis Protein synthesis was analysed by incorporation of [35S]methionine (Amersham Pharmacia Biotech) into pro-teins as described earlier [9] Propro-teins were precipitated after
5 h of incubation at 37C in aerobic conditions in LB broth The final concentration used for [35S]methionine was 0.1 mM at 100 lCi The precipitate was collected onto GF/C filters (0.45 lm), washed and radioactivity was counted in a scintillation counter Protein synthesis was detected in duplicate samples and results were expressed as nmol of [35S]methionine incorporated per lg of protein The
Trang 3detection limit of this method was 0.01 nmol [35
S]methio-nine per lg protein, corresponding to protein synthesis of
about 106CFUÆmL)1 The maximal deviation from the
mean of two independent experiments was calculated
Glucose uptake
Duplicated samples of 2 mL of UV-treated and untreated
cell suspension were incubated with or without 440 U of
catalase (220 UÆmL)1) After 15 min of incubation at room
temperature, 5 mMof glucose (final concentration) spiked
with [14C]glucose (10 lCi in the 2 mL of mix) (Amersham
International) were added The reaction mixtures were
incubated in aerobic conditions at 37C with shaking
(180 r.p.m) Aliquots were taken at different times,
deposi-ted on GF/C filters (pore size, 0.45 lm; 2.5 cm diameter;
Whatman, Maidstone, England) and then washed with
phosphate buffer to remove nonincorporated [14C]glucose
Each filter was dried and radioactivity was measured in a
scintillation counter Glucose uptake with catalase
previ-ously inactivated in water at 100C for 30 min was used as
a negative control In order to avoid precipitation of
heat-inactivated catalase in negative control experiments,
phos-phate buffer without NaCl was used The results obtained
were expressed as nmol [14C]glucose transported per lg of
protein The detection limit of this method was 0.5 nmol
[14C]glucose per lg protein corresponding to glucose uptake
of about 107CFUÆmL)1 The maximal deviation from the
mean of two independent experiments was calculated
Metabolic flux by13C NMR spectroscopy
As in the case of glucose uptake, cell suspensions were
incubated with or without catalase Here, 13C glucose
(Leman, St Quentin en Yvelines, France) labelled at C1 or
C6 was used and the reaction mixtures were incubated 4 h
in aerobic or anaerobic conditions When glucose, labelled
isotopically either in position C1 or C6, is added to bacterial
suspension, the amount of label introduced in acetate C2
depends on the activity of the pentose phosphate (PP)
pathway The equations used for estimating the relative
activities of the PP or Embden–Meyerhof (EM) pathway
were: y) x ¼ PP, x ¼ EM, where x was the C2
enrich-ment of the acetate measured from [1-13C]glucose and y the
C2 enrichment of the acetate measured from [6-13C]glucose
Perchloric acid extraction was performed to prevent a
possible alteration of the secretion of the metabolites due to
the UV-treatment The reaction was stopped by addition of
240 lL of perchloric acid at 4C The samples were
vortexed for 2 min, placed in ice for 15 min, vortexed again
for 2 min and finally centrifuged at room temperature at
8000 g for 15 min Acid extracts were neutralized to pH 7
with NaOH and stored at)20 C until NMR analysis All
NMR data were recorded at 303Kon a Bruker Avance 400
spectrometer using a 10-mm broad-band probe Neutralized
extracts were introduced in a 8-mm NMR tube, itself
inserted in a 10-mm NMR tube containing D2O.13C NMR
spectra recorded at 100.13 MHz were acquired during 1 h
(2400 scans) with a composite pulse decoupling
Exponen-tial filtering of 3 Hz was applied prior to Fourier
transfor-mation Chemical shifts were referred to the a-C1 resonance
of -glucose (93.1 p.p.m) The acetate concentration and
other metabolites formed (glucose, lactate, ethanol) were determined by NMR analysis and enzymatic assays (Boeh-ringer, Mannheim, Germany) as described previously [19]
Results
Loss of culturability after UV-treatment The physiological state of nonculturable E coli cells obtained by irradiation with a low (UV1) and a high (UV2) UV dose was examined Immediately following the
UV treatment, no decrease in the total number of cells was observed The total cell count was 2.8· 108 cellsÆmL)1 (± 4%) for untreated and UV1- or UV2-treated cells After both UV treatments the great majority of the population ( 108cellsÆmL)1) became nonculturable on LB agar plates while a minor percentage remained culturable (0.001– 0.0001%) However, no interference from these few cultur-able cells (UV survivors) was observed in further experi-ments because their number remained much lower than the detection limit of the method used Loss of culturability on nutrient media could be explained by direct and indirect damage to nucleic acids produced by UV radiation [20] Direct effects of UV radiation at 254 nm on nucleic acids include, for example, photodimerization between adjacent pyrimidine bases Indirect effects result when reactive oxygen species such as hydrogen peroxide are generated They also react with DNA, damaging bases, breaking strands and cross-linking DNA and protein [2] In our experiments, catalase and superoxide dismutase were added
to the medium to enhance culturability by protecting against the effects of free radicals However, no increase in colony count on LB agar plates of UV1- and UV2-treated cells previously incubated in phosphate buffer containing
220 UÆmL)1of catalase for 2 h, 4 h, or 24 h was observed Furthermore, neither the addition of 2000 U catalase or both catalase and superoxide dismutase on LB agar plates nor incubation in anaerobic conditions reversed this result After prolonged incubation (5 days) no further colony development could be observed
During the first 24 h of incubation time without nutrients after UV1-irradiation, the proportion of total and UV-survivor cells remained constant However, in the case of cells treated with the UV2 dose, a decrease of about 30% in the original total cell number indicated the existence of cell lysis This decrease in the total cell number was followed by
a small increase in the number of culturable cells, which, after 24 h of incubation, reached almost 0.1% of the initial value The regrowth is most probably explained by growth
of the minor percentage of UV-survivors cells at the expense
of nutrients liberated by UV2 lysed cells Cell lysis could be explained by loss of the ability of UV2-treated cells to modify their autolysins Inhibition of murein synthesis and loss of the electrical or pH gradient of cellular membranes have been described as ways to trigger lysis due to the uncontrolled autolytic action of murein hydrolases [21,22] Response to nutrients
Epifluorescence microscopy was used to determine whether, immediately after the UV-treatment, cells that lost cultura-bility had the acultura-bility to produce cell elongation with the
Trang 4DVC method (incubation with nutrients and nalidixic acid).
After DVC of UV1-treated cells, DVC positive cells that
exceeded at least twice the mean length of E coli K-12 were
observed (Fig 1, B1 and B2) Nevertheless, a few slightly
elongated cells were observed even without nalidixic
addi-tion However, this change in cell size was not detectable or
quantifiable even after analysis of a great number of cells by
flow cytometry (data not shown) To determine whether
slightly elongated or nonelongated UV1-treated cells
responded to nutrient addition, an improved DVC method
(qDVC) was used With this method, substrate-responsive
cells were selectively lysed by spheroplast formation caused
by incubation with nutrients, nalidixic acid and glycine It is
known that glycine interferes with several steps in
pepti-doglycan synthesis for bacterial cell wall formation [23], and
this effect leads to swollen cells with a very loose cell wall
The substrate-responsive cells were lysed easily by a freezing
treatment in liquid nitrogen and then thawed at room
temperature With this method, the proportions of substrate
responsive cells obtained were 90% for untreated and 60%
for UV1-treated cells Cell lysis was not observed in the
negative controls without glycine addition When DVC
(Fig 1, C1 and C2) and qDVC were performed using
UV2-treated cells, substrate responsive cells were not detected
With these cells, no elongated cells were detected after DVC,
and after qDVC cell lysis was detected in both samples, with
or without glycine addition
To obtain more evidence of the response to nutrients, the
incorporation of [14C]glucose into cells and the effect of
exogenous catalase on glucose uptake were studied (Fig 2)
For untreated cells, glucose incorporation in the absence of
catalase reached a steady state level of about 3.6 lmol
[14C]glucose per lg protein after 4 h of incubation
UV1-treated cells incorporated 2.5 lmol [14C]glucose per lg
protein corresponding to 69% of the glucose incorporated
by untreated cells However, for UV2-treated cells, a large
decrease in the maximal glucose incorporation (0.6 lmol [14C]glucose per lg protein) was observed, corresponding
to only 17% of the glucose incorporated by untreated cells (Fig 2A) When the same experiments were carried out in the presence of catalase, an increase in glucose uptake of about 60% was observed (Fig 2B) in untreated and UV1 and UV2-treated cells For UV2-treated cells this increase was observed only during the first 4 h of glucose incorpor-ation After this time, glucose uptake with or without catalase addition decreased (data not shown), undoubtedly explained by the beginning of cell lysis described above In all cases, no increase in glucose uptake was observed when experiments were carried out with catalase previously inactivated at 100C In nongrowth conditions, the
Fig 1 Visualization of cells Visualization by fluorescent in situ hybridization of untreated cells (A), UV1-treated cells (B) and UV2-treated cells (C) before (A1, B1, C1) and after DVC method (A2, B2, C2).
Fig 2 Glucose uptake Glucose uptake in aerobic conditions (A), non irradiated cells (•, NI), UV1-treated cells (j, UV1) and UV2-treated cells (m, UV2) Glucose uptake in aerobic conditions after 4 h of incubation with or without exogenous catalase (B) The detection limit
of this method was 0.5 nmol [ 14 C] glucose per g protein corresponding
to glucose uptake of about 107CFUÆml)1(10% of the initial number
of cells).
Trang 5imbalance during glucose uptake between cell metabolism
and the arrest of cell division could be favorable to peroxide
generation and accumulation In E coli, peroxide arises
primarily from the auto-oxidation of components of its
respiratory chain [24], and the presence of peroxide induces
membrane damage [25] Thus, prevention by exogenus
catalase of peroxide damage could explain the observed
increase in glucose uptake However, this effect was
observed indifferently in untreated and both UV1- and
UV2- treated cells, showing no relation with the degree of
UV-damage
To obtain more information on the physiological state of
UV-treated cells, protein synthesis was analysed (Fig 3)
After 1.5 h of incubation with [35S]methionine, UV1-treated
cells synthesized less protein than untreated cells Then,
both untreated and UV1-treated cells reached a maximal
incorporation of about 1.5 nmol of [35S]methionine per lg
of protein As expected, no [35S]methionine incorporation in
proteins for UV2-treated cells was detected
Glucose metabolism under different aeration conditions
The capacity of E coli cells to metabolize glucose was
investigated in whole cells using13C-NMR spectroscopy
13C-NMR studies were performed in untreated and
UV-treated cells incubated for 4 h in aerobic and anaerobic
conditions and the concentrations of fermentative products
were measured by enzymatic assays Results with enriched
[1-13C]glucose are shown in Fig 4 In anaerobic conditions
for untreated and UV1-treated cells, a similar NMR
spectrum was obtained Acetate (A), lactate (L) and ethanol
(E) were the main products formed, at levels of about 20, 90
and 10 mol per 100 mol of metabolized glucose, respectively
However, for UV2-treated cells, less glucose was consumed,
a lower concentration of lactate and acetate was observed
and no ethanol was detected (Fig 4) The fact that ethanol is
not detected in UV2-treated cells could be explained by the
reduced glucose consumption or most probably by the
incapacity of these cells to synthesize proteins In anaerobic
conditions, only cells that can synthesize the pyruvate
formate lyase de novo can form ethanol [26] In aerobic
conditions, acetate was the only product detected in cellular extracts, the concentration for untreated and UV1-treated cells being about 90 mol per 100 mol of metabolized glucose For UV2-treated cells, 70 mol of acetate per 100 mol of metabolized glucose were detected
In E coli, glucose is metabolized via the EM and PP pathways [27,28] In order to determine whether UV-treated cells incubated in different aeration conditions were able to modify their metabolic flux of glucose, the activities of the
EM and PP pathways were studied Glucose metabolism through these two pathways was quantified separately by using glucose substrates with a13C label at different carbon atoms An easy and versatile method to determinate the acetate concentration by NMR after incubation with [6-13C]glucose or [1-13C]glucose was used to quantify separately the EM and PP competing pathway contribution
If glucose labelled isotopically either in C1 or C6 positions is added to bacterial suspensions, the amount of label introduced in acetate C2 will depend on the activity of the PP pathway In fact, when [1-13C]glucose is used as the carbon source, part of the13C label is lost as CO2 in the phosphogluconate dehydrogenase step of the PP pathway, whereas the other part of the13C label is incorporated into acetate C2 via the EM pathway On the other hand, when [6-13C]glucose is used as the carbon source, all the13C label
is incorporated into acetate C2 via the EM and PP pathways [29] Even though the described procedure is a simplified flux estimation because other possible CO2-liberating reac-tions are neglected [30] it allowed a first estimation of the flux differences between untreated and UV-treated cells For untreated and both UV1- and UV2-treated cells, the relative activities of the EM and PP pathways in anaerobic conditions were about 91% and 9%, respectively (Table 1)
In untreated cells, the initial rate of glucose consumption was 7 nmol per lg protein per min and a similar rate was observed in UV1- and UV2-treated cells Considering aerobic conditions, only untreated and UV1-treated cells had the capacity to modify metabolic flux through both pathways, the relative activity of the EM and PP pathways
Fig 3 Protein synthesis Protein synthesis of untreated cells (•),
UV1-treated cells (j), UV2-treated cells (m), detected by
incorpor-ation of [35S]methionine The detection limit of this method was
0.01 nmol of [ 35 S]methionine per g protein corresponding to protein
synthesis of about 10 6 CFUÆml)1(1% of the initial number of cells).
Fig 4 13 C-NMR spectra 13 C-NMR spectra of untreated, UV1- and UV2-treated cells after 4 h of incubation with [1–13C] glucose in anaerobic conditions The glucose anomers, a and b are visible as well
as three end products of glucose metabolism, acetate (A); lactate (L) and ethanol (E).
Trang 6being 44% and 56% (Table 1), respectively In these
cells, an increase of at least fourfold in the rate of glucose
consumption was observed (about 30 nmol per lg protein
per min) In contrast, UV2-treated cells were unable to
respond to variations in aeration conditions These cells
showed a similar metabolic flux through both pathways
and rate of glucose consumption in aerobic and anaerobic
conditions In this study, the flux estimation was
deter-mined by MNR in cells incubated in nongrowing
condi-tions Nevertheless, the same method applied to E coli
grown anaerobically gives similar flux values (22% by the
PP pathway) [31] Using more comprehensive methods
such as GC-MS, it has been confirmed recently, that in
growing cells, the oxidative PP pathway is still active
under anaerobic conditions and decreases with decreasing
oxygen availability [32]
As described above, during glucose incorporation in
nongrowth conditions, peroxide was generated in cells It
was expected that both the EM and PP pathway activity
would be affected by peroxide, which freely diffuses into
cells, harming cell proteins Furthermore, in vitro assays
showed that peroxide inhibited the activity of several E coli
K-12 enzymes such as phosphogluconate dehydrogenase,
alcohol dehydrogenase, lactate dehydrogenase and acetate
kinase (data not shown) However, we obtained the same
flux through the EM and PP pathways in experiments where
peroxide was degraded or not through the addition of
exogenous catalase This result, along with the evidence of
retention of protein synthesis described above, might be
indicative of preservation in UV1-treated nonculturable
cells of intracellular catalase activity, which prevents
intra-cellular damage to cells Indeed, a homeostatic regulation of
intracellular hydrogen peroxide concentration by the
pro-duction of intracellular catalase, but in culturable E coli
cells, has already been observed [33]
Discussion
Cells were defined operationally as alive or dead depending
on the method used to determine cell viability For example,
using the capacity of cell division or elongation as a criterion
for bacterial life, cells treated with UV1 and UV2 doses
could be diagnosed as dead cells In contrast, if the capacity
to transport or metabolize glucose is used as a criterion of
bacterial life, cells treated with UV1 and UV2 doses could
be diagnosed as living cells This study suggests that global
information on intracellular stability (protein synthesis,
metabolic flux) is needed to define with less ambiguity the
physiological state of nonculturable cells However, in the
absence of consensus on the definition of bacteria death
(independent of culturability), UV1-treated cells could be diagnosed as simply in a metabolically active state and not
as living cells After addition of nutrients, nonculturable cells obtained by UV1 irradiation maintained the capacity
to synthesize proteins and peptidoglycan They also retained both a high level of glucose transport and the capacity to metabolize glucose at the same rates as those of nontreated cells Moreover, UV1-treated cells retained the capacity to modify their metabolic flux through the EM and PP pathways after variation of aeration conditions On the other hand, nonculturable cells obtained by irradiation with the UV2 dose were clearly in a metabolically inactive state UV2-treated cells not only showed a gradual loss of cell integrity, they also lost the capacity to respond to nutrient addition by cell elongation or protein synthesis and the capacity to modify their metabolic flux in glucose metabo-lism after variation of aeration conditions UV2-treated cells retained only residual metabolic activity and showed several levels of metabolic imbalance
To clarify the medical significance (when pathogenic) of bacteria that lose culturability, further studies should be performed to examine the persistence of these active cells and their capacity to repair their damage, to produce important metabolites (e.g toxins) and restart cell division
Acknowledgement
Aquabiolab is supported by Anjou Recherche/Vivendi Water Aqu-abiolab supported the PhD scholarship of Andrea Villarino We acknowledge the technical assistance of Marie Christine Wagner, Analytic and Preparative Cytometry Service, Institut Pasteur.
References
1 Lazarova, V., Savoye, P., Janex, M.L., Blatchley, E.R & Pom-mepuy, M (1999) Advanced wastewater disinfection technologies: State of the art and perspectives Water Sci Technol 40, 203–213.
2 Russell, A.D (1998) Preservation and Sterilization In Principles and Practice of Disinfection (Rusell, A.D., Hugo, W.B & Ayliffe, G.A.J., eds) pp 630 Blackwell Science, Oxford.
3 Sommer, R., Haider, T., Cabaj, A., Pribil, W & Lhotsky, M (1998) Time dose reciprocity in UV disinfection of water Water Sci Technol 38, 145–150.
4 Bruch, C.W & Bruch, M.K (1971) Sterilization In Husa’s Pharmaceutical Dispensing (Martin, E.W., ed.), pp 592–623 Mack Publishing, Easton, PA.
5 Postgate, J.R., Crumpton, J.E & Hunter, J.R (1961) The measurement of bacteria viabilities by slide culture J General Microbiol 24, 15–24.
6 Wilber, L.A & Oliver, J.D (2000) Ultraviolet light induces the VBNC state in Salmonella typhimurium and Escherichia coli Abstr Gen Meeting Am Soc Microbiol., p 400 Los Angeles, CA, USA.
7 Muela, A., Garcia-Bringas, J.M., Arana, I & Barcina, I (2000) The effect of simulated solar radiation on Escherichia coli: the relative roles of UV-B, UV-A, and photosynthetically active radiation Microb Ecol 39, 65–71.
8 Caro, A., Got, P., Lesne, J., Binard, S & Baleux, B (1999) Via-bility and virulence of experimentally stressed nonculturable Sal-monella typhimurium Appl Environ Microbiol 65, 3229–3232.
9 Villarino, A., Bouvet, O.M.M., Regnault, B., Martin-Delautre, S.
& Grimont, P.A.D (2000) Exploring the frontier between life and death in Escherichia coli: Evaluation of different viability markers
in live and heat- or UV-killed cells Res Microbiol 151, 755–768.
Table 1 Relative activity (%) of the Embden–Meyerhof (EM) and
pentose phosphate (PP) pathways of E coli K-12 cells Results are the
mean of at least two independent experiments.
Trang 710 Choi, J.W., Sherr, E.B & Sherr, B.F (1996) Relation between
presence-absence of a visible nucleoid and metabolic activity in
bacterioplankton cells Limnol Oceanogr 41, 1161–1168.
11 Kell, D.B., Kaprelyants, A.S., Weichart, D.H., Harwood, C.R &
Barer, M.R (1998) Viability and activity in readily culturable
bacteria: a review and discussion of the practical issues Antonie
Leeuwenhoek 73, 169–187.
12 Appleyard, R & K (1954) Segregation of new lysogenic types
during growth of a doubly lysogenic strain derived from
Escher-ichia coli K12 Genetics 39, 440–452.
13 Maniatis, T., Fritsch, E.F & Sambrook, J (1982) Molecular
Cloning: A laboratory manual Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
14 Kogure, K., Simidu, U & Taga, N (1979) A tentative direct
microscopic method for counting living marine bacteria Can J.
Microbiol 25, 415–420.
15 Yokomaku, D., Yamaguchi, N & Nasu, M (2000) Improved
direct viable count procedure for quantitative estimation of
bac-terial viability in freshwater environments Appl Environ
Micro-biol 66, 5544–5548.
16 Regnault, B., Martin-Delautre, S., Lejay-Collin, M., Lefevre, M.
& Grimont, P.A.D (2000) Oligonucleotide probe for the
visuali-zation of Escherichia coli/Escherichia fergusonii cells by in situ
hybridization: Specificity and potential applications Res
Micro-biol 151, 521–533.
17 Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W.,
Devere-ux, R & Stahl, D.A (1990) Combination of 16S ribosomal
RNA-targeted oligonucleotide probes with flow cytometry for analyzing
mixed microbial populations Appl Environ Microbiol 56,
1919–1925.
18 Davey, H.M & Kell, D.B (1996) Flow cytometry and cell sorting
of heterogeneous microbial populations: The importance of
single-cell analyses Microbiol Rev 60, 641–696.
19 Rager, M.-N., Binet, M & Bouvet, O.M.M (1999)31P and13C
nuclear magnetic resonance studies of metabolic pathways in
Pasteurella multocida: Characterization of a new
mannitol-producing metabolic pathway Eur J Biochem 263, 695–701.
20 Miller, R.V., Jeffrey, W., Mitchell, D & Elasri, M (1999)
Bac-terial responses to ultraviolet light ASM News 56, 535–541.
21 Holtje, J.V (1995) From growth to autolysis: the murein
hydro-lases in Escherichia coli Arch Microbiol 164, 243–254.
22 Jolliffe, L.K., Doyle, R.J & Streips, U.N (1981) The energized membrane and cellular autolysis in Bacillus subtilis Cell 25, 753–764.
23 Hammes, W., Schleifer, K.H & Kandler, O (1973) Mode of action of glycine on the biosynthesis of peptidoglycan J Bacteriol.
116, 1029–1053.
24 Messner, K.R & Imlay, J.A (1999) The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli J Biol Chem 274, 10119–10128.
25 Hoshino, N., Kimura, T., Yamaji, A & Ando, T (1999) Damage
to the cytoplasmic membrane of Escherichia coli by catechin-copper (II) complexes Free Rad Biol Med 27, 1245–1250.
26 Gottschalk, G., ed (1985) Bacterial Metabolism Springer-Verlag, New York.
27 Neidhardt, F.C., Ingraham, J.L & Schaechter, M (1990) Bio-synthesis and fueling In Physiology of the Bacterial Cell: a Molecular Approach (Neidhardt, F.C., Ingraham, J.L & Schaechter, M., eds), pp 133–173 Sinauer Associates, Inc., MA.
28 Nystrom, T., Larsson, C & Gustafsson, L (1996) Bacterial defense against aging: Role of the Escherichia coli ArcA regulator
in gene expression, readjusted energy flux and survival during stasis EMBO J 15, 3219–3228.
29 Bouvet, O.M.M & Rager, M.-N (2000) Sugar transport and metabolism in fermentative bacteria In NMR in Microbiology: Theory and Applications (Barbotin, J.-N & Portais, J.-C., eds), pp 349–361 Horizon Scientific Press, England.
30 Sauer U., Lasko, D.R., Fiaux, J., Hochuli, M., Glaser, R., Szy-persky, T., Wu¨thrich, K & Bailey, J.E (1999) Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism J Bacteriol 181, 6679–6688.
31 Ogino, T., Arata, Y & Fujiwara, S (1980) Proton correlation nuclear magnetic resonance study of metabolic regulations and pyruvate transport in anaerobic Escherichia coli cells Biochemistry
19, 3684–3691.
32 Fisher, E & Sauer, U (2003) Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS Eur J Biochem 270, 880–891.
33 Gonzalez-Flecha, B & Demple, B (1997) Homeostatic regulation
of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli J Bacteriol 179, 382–388.