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LONGITUDINAL EVALUATION OF THE EFFICACY OF HEAT TREATMENT 1

PROCEDURES AGAINST LEGIONELLA SPP. IN HOSPITAL WATER SYSTEMS 2

BY USING FLOW CYTOMETRIC ASSAY 3

4

RUNNING TITLE: Legionella heat treatment evaluated by flow cytometry 5

6

Severine Allegra, Florence Grattard, Françoise Girardot, Serge Riffard, 7

Bruno Pozzetto and Philippe Berthelot* 8

9

Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP), EA 3064 10

Université de Lyon, Université Jean Monnet et CHU de Saint-Etienne, 11

42023 Saint-Etienne, France. 12

13

14

* Corresponding author: 15

Pr. Philippe Berthelot, MD, M.P.H., PhD 16

Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP), EA 3064 17

Unité d’Hygiène, Centre hospitalo-universitaire de Saint-Etienne, 42055, Saint-18

Etienne cedex 02, France 19

Telephone number: + 33 (0) 4 77 82 88 26 20

Fax number: + 33 (0) 4 77 12 04 39 21

Email address: [email protected] 22

23

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02225-10 AEM Accepts, published online ahead of print on 23 December 2010

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ABSTRACT 1

2

Legionella spp. is frequently isolated in hospital water systems. Heat shock (30 min 3

at 70°C) is recommended by the World Health Organization to control its 4

multiplication. The aim of the study was to evaluate retrospectively the efficacy of 5

heat treatments by using a flow cytometry assay (FCA) able to identify viable but non 6

culturable (VBNC) cells. The study included Legionella strains (L. pneumophila, 3 7

clusters; L. anisa, 1) isolated from four hot water circuits of different hospital buildings 8

in Saint-Etienne, France, during a 20-year prospective surveillance. The strains 9

recovered from the different circuits were non epidemiologically-related but the 10

strains isolated within a same circuit over time exhibited an identical genotypic 11

profile. After an in vitro treatment of 30 minutes at 70°C, the mean percentage of 12

viable cells and VBNC varied from 4.6 to 71.7. The in vitro differences in heat 13

sensitivity were in agreement with the observed efficacy of heating preventive and 14

corrective measures used to control the water contamination. These results suggest 15

that Legionella strains can become heat-resistant after heating treatments for a long 16

time and that flow cytometry could be helpful to check the efficacy of heat treatments 17

on Legionella spp. and to optimize the decontamination processes applied to water 18

systems for the control of Legionella proliferation. 19

20

Key words: Legionella, viable but not culturable cells, culture, flow cytometry, 21

disinfection treatment, water system. 22


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INTRODUCTION 1

2

Legionella are widespread in natural and man-made aquatic habitats. Approximately 3

one-third of the Legionella species have been associated with a severe pneumonia in 4

humans, the Legionnaires’ disease (13, 32). Sources of contamination are aerosols 5

from showerheads, spas, air-cooling towers or other systems distributing hot water. 6

In order to prevent outbreaks, surveillance of Legionella environmental contamination 7

is recommended in hot sanitary water systems of collective settings such as 8

hospitals, hotels or thermal institutes (2). In France, environmental surveillance of hot 9

water is mandatory for hospitals; to minimize the risk of Legionella infection 10

recommended concentrations of Legionella pneumophila must be at least under 11

1000 colony-forming units per liter (CFU/L) and under the detection threshold (<250 12

CFU/L) for immunocompromised patients. A disinfection is required for bacterial load 13

higher than 10,000 CFU/L (12, 28). Many disinfection methods have been proposed 14

to control Legionella proliferation in hot water systems, including thermal treatments 15

(delivery of water at 55°C or heat shocks at 70°C), and chemical procedures 16

(continuous or shock chlorination, use of continuous chlorine dioxide, 17

monochloramine, ozone, aldehydes) (19). However, these disinfection procedures 18

performed in hospital hot water systems (6, 8, 33) have often a short term efficacy, 19

the re-colonization occurring after only some weeks or months. Indeed, L. 20

pneumophila cell populations have been shown to survive as free organisms for long 21

periods by maintaining metabolic activity but temporarily losing culturability under 22

strict environments and requiring resuscitation by ingestion by amoebas (14, 25, 34). 23

In a previous work using a flow cytometric assay (FCA) (1), we confirmed the 24


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existence of viable but not culturable (VBNC) Legionella cells (3, 26) in 1

environmental samples. 2

The aim of this study was to evaluate the adding value of FCA for estimating the 3

efficacy of heat treatment procedures used for Legionella disinfection in water 4

systems thanks to strains collected prospectively during environmental surveillance 5

of the water circuits of a University hospital. For each collected strain, FCA profiles 6

were obtained before and after different times of heat-shock at 70°C. The results 7

were compared to the decontamination procedures applied to the water circuits from 8

which the strains had been recovered. 9

10

11

MATERIALS AND METHODS 12

13

Setting 14

The University hospital of Saint-Etienne is composed of several buildings located on 15

two sites and supplied with hot water through independent circuits. From 1992 to 16

1994, a nosocomial legionellosis outbreak (4, 16) had occurred due to water circuit 17

contamination. At that time, there was no mandatory environmental surveillance of 18

hospital hot water system and no disinfection procedure. In 1995, an environmental 19

surveillance procedure of the whole water system was set up. Sites and frequency of 20

hot water sampling were defined in each building by the staff of the infection control 21

unit and engineers, according to the complexity of the water system and to the 22

potential exposure of patients at risk. Hot water samples were collected from 23

showers or hot tap water. The three water circuits of the University hospital were 24

designated A to C. In 2005, a new hospital located on the same site was opened; its 25


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water circuit was designated as D. The pipes were made of copper for circuits A to C 1

and mainly of cross-linked polyethylene and chloride polyvinyl for circuit D. 2

3

Legionella strains recovery 4

Thirty nine Legionella strains isolated from 1992 to 2010 and stored at -80°C in 5

cryobank tubes (Mast Diagnostic, Amiens, France) were used for this study. They 6

were isolated and identified according to the French AFNOR norm NF T90-431 (18, 7

23). Legionella colonies from frozen samples were recovered by plating on BCYEα 8

agar medium (buffered activated charcoal and yeast extract, Legionella CYE and 9

SR0110 supplement; Oxoid, Dardilly, France) and after an incubation for 3 days at 36 10

± 2°C before experiment. 11

12

AP-PCR typing 13

The genomic diversity of the L. pneumophila serogroup 1 strains and of L. anisa was 14

analyzed by arbitrarily primed PCR (AP-PCR) using Eric 2 primer: 5’-15

AAGTAAGTGACTGGGGTGAGC-3’ and primer G: 5’-GGTGGTGGCT-3’, as 16

previously described (16). 17

18

Preparation of calibrated suspensions of Legionella 19

All suspensions were prepared by suspending Legionella colonies into sterile normal 20

saline (NaCl 0.9%) to get an optical density of 0.2 at 600nm (Biomate TM3, Avantec, 21

Illkirch, France), which corresponds to a final concentration of 108 CFU/mL. 22

23

24

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In vitro heat shock treatments 1

Calibrated suspensions of strains collected were diluted into sterile normal saline to 2

106 CFU/mL and then incubated in a water bath set at 70°C for 0, 10, 30 and 60 3

minutes. 4

5

Flow cytometric assay 6

For FCA, each strain was used at a concentration of 105 CFU/mL. FCA profiles were 7

obtained using a combination of two fluorescent dyes staining nucleic acids, Syto9 8

for cells with intact membrane and propidium iodide (PI) for cells with damaged 9

membrane, as previously described (1). Flow cytometric measurements were 10

performed by using a BD FACSCaliburTM (Becton Dickinson Biosciences, Le Pont-11

de-Claix, France) equipped with an air-cooled argon laser (488 nm emission, 20mW). 12

The green fluorescent emission from Syto9 was collected in FL1 channel (530/30 13

nm) and the red fluorescence from PI in FL3 channel (>670 nm). A threshold was 14

applied on the FL1 channel to eliminate background signal. Analyses were performed 15

at a low flow rate setting. Results were analyzed with the Cell Quest ProTM software 16

(Becton Dickinson Biosciences) as previously described (1). 17

18

RESULTS 19

20

Presentation of the different water circuits contaminated by Legionella 21

Figure 1 illustrates the follow-up of Legionella contamination of the four water circuits 22

taken into consideration in this study and the main control measures that were 23

applied through time. Circuits A to C were found contaminated by Legionella 24

pneumophila serogroup 1 whereas circuit D was contaminated with L. anisa. Circuit 25


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A illustrates the efficacy of punctual heat shock treatments leading to the drastic 1

reduction of Legionella load over time. At the opposite, circuit C illustrates the 2

inability of temperature rise to control the Legionella reservoir, leading to the set-up 3

of a chlorine pump in 2006. By comparison to the two previous circuits, circuit B 4

represents an intermediate situation with an initial control of Legionella growth by 5

combination of heat shock and temperature rise measures, followed by the 6

resurgence of Legionella contamination after 2000 leading in 2005 to the set-up of a 7

chlorine pump. Circuit D, contaminated by L. anisa, was directly treated by chlorine 8

disinfection because the pipe material including polyvinyl chloride did not support 9

high temperature. 10

11

AP-PCR and FCA patterns of Legionella strains 12

Legionella strains isolated from the four water circuits described above and illustrated 13

by stars in Figure 1 were used to study their temperature sensitivity through time by 14

using FCA. First, in order to verify their clonal character, they were all tested by AP-15

PCR: strains from a same water circuit were shown to share the same profile 16

whereas each circuit was contaminated by a different clone (Figure 2). These results 17

demonstrate the persistence over time of the same Legionella strain in each 18

independent circuit, even for a long period of time (at least 16 years for circuit B). For 19

each strain displayed in Figure 1, the respective percentages of viable and culturable 20

(VC), viable but not culturable (VBNC) and dead (D) cells were then evaluated by 21

FCA. The cytograms of representative strains are displayed in Figure 3. Interestingly, 22

as shown for AP-PCR profiles, the distribution among the three cell categories (VC, 23

VBNC and D) was very similar between strains isolated over time from a same circuit 24

but different from a circuit to another. 25


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FCA analysis of Legionella susceptibility to temperature 1

In order to analyze the temperature susceptibility of the Legionella strains shown in 2

Figure 1, each of them was submitted to a heat treatment at 70°C during 0, 10, 30 3

and 60 minutes. The respective percentages of VC, VBNC and D cells were then 4

determined by FCA. Figure 4 (individual patterns of each strain after 30 minutes at 5

70°C) and Figure 5 (mean kinetic of inactivation at 70°C through time) illustrate the 6

temperature susceptibility of the L. pneumophila strains isolated from circuits A to C. 7

Cytometric analyses clearly discriminate two main profiles, a profile of high 8

susceptibility to heating (more than 75% of dead cells at 30 min) represented by all 9

the strains of circuit A and the first 5 strains of circuit B (cluster B1), and a profile of 10

resistance to heating (less than 50% of dead cells at 30 min) represented by all the 11

strains of circuit C and the 7 last strains of circuit B (cluster B2). It is worthwhile to 12

note that, despite identical AP-PCR patterns (Figure 2) and similar FCA profile before 13

heating (Figure 3) over all the study period, the reservoir of L. pneumophila from 14

circuit B, which was submitted regularly to heating treatment, evolved from 15

susceptible to resistant to heating through time. As shown in Figure 4, the change 16

occurred between 1994 and 2000; as no strain was kept frozen in the meantime, it 17

was not possible to determine more precisely the moment at which this evolution 18

took place. Table 1 synthesizes the pooled results of heat susceptibility of strains 19

from each circuit before and after treatment at 70°C. As determined by FCA, the 20

mean percentage of viable cells after 30 minutes at 70°C discriminates well the two 21

profiles and is close to that obtained for viable cells after a heating time of 60 22

minutes. L. anisa strains recovered from circuit D exhibited a highly susceptible 23

profile to heating (Table 1). 24

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DISCUSSION 1

2

This study was conducted on water circuits from two French hospitals that were 3

found chronically-contaminated with Legionella species, from 5 years for circuit D to 4

at least 18 years for circuit B (Figure 1). The environmental surveillance was started 5

following the occurrence of a nosocomial outbreak (4, 16), a few years before it 6

became recommended by the French legislation. The main hygienic measures taken 7

to circumvent the contamination are depicted in Figure 1. No further cases of 8

hospital-acquired legionellosis have been recorded for a period of 18 years in our 9

setting. These results plead for the usefulness of environmental surveillance, as 10

recommended by several European guidelines (5, 7, 12), and in accordance with the 11

results of the Allengheny County Health Department in the USA (11, 31). Convinced 12

of the efficacy of environmental monitoring, we undertook the present study to 13

evaluate the interest of FCA as a refined tool of measure of Legionella susceptibility 14

to heating in the context of our hospital setting. 15

16

FCA was first tested to analyze the relative distribution of cells of different viability 17

within a same Legionella strain population in the absence of any treatment. It was 18

concluded that FCA profiles were relatively similar for different strains contaminating 19

a same circuit but different for strains isolated from distinct circuits (Figure 3). 20

Interestingly, these results were in accordance with AP-PCR typing data depicted in 21

Figure 2, using two different primers. By the way, the clonal character of Legionella 22

strains contaminating a same circuit has already been shown by previous works (10, 23

24, 27, 30). The present study documents a water circuit contamination by the same 24

strain of Legionella for a period of up to 18 years (circuit B). 25


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The main objective of the study was to evaluate the ability of FCA to measure the 1

susceptibility to heating treatment of Legionella strains recovered from the 2

environment. The use of FCA was based on the fact that this technique is a 3

performing tool to demonstrate the existence of VBNC in Legionella populations (1). 4

By now, raising the temperature is considered as one of the best way to control the 5

contamination of water circuits by Legionella (20, 21, 35) and constitutes the 6

rationale for several guidelines. The minimum temperature for Legionella thermal 7

disinfection is 60°C since the time required to obtain 1-log kill (90% reduction) at 8

45°C, 50°C, 60°C, and 70°C was 2500, 380, <5, and <1 min, respectively (17, 19-9

21). By contrast, one of the major findings of the present study was that, by using 10

FCA, some strains of Legionella submitted in the environment to superheating for a 11

long time were shown to develop a resistance to high temperature. This phenomenon 12

was demonstrated by the high proportion of culturable and not culturable viable cells 13

still present after a 30 min treatment at 70°C (Figures 4 and 5, Table 1). The 14

percentage of VBNC of Legionella in environmental samples is likely to be 15

associated with variations of biotic or abiotic factors affecting the ecosystem in which 16

Legionella agents proliferate (1, 14, 26, 33). 17

A further factor that fosters the survival and dissemination of Legionella in aquatic 18

environments is the biofilm (22, 29). Even if it is not clear whether the pipe material 19

(37) or the type of disinfection influence the development of biofilm, it has been 20

demonstrated that the presence of biofilm reduces the efficacy of disinfection 21

treatments (36). For example, incorporation of natural non cultivable L. pneumophila 22

into potable water biofilms provides a protective niche against chlorination stress 23

(15). Moreover, if Legionella are present at high density, they may communicate in a 24

way that enables them to better survive within a stressful environment (25, 38). In 25


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close relation to our study and by using a similar approach, Chang et al. 1

demonstrated recently that starvation enhances significantly the resistance of 2

Legionella to superheating or chlorination (9). These authors postulate that 3

stabilization of cell membrane and/or induction of proteins and other gene products 4

may explain the resistance of starved strains to heat stress (9). Our results obtained 5

by using FCA illustrate for the first time the ability of repetitive heat shocks to 6

generate heat-resistant Legionella strains in the environment. The intensity of the 7

heating treatment seems to act as a factor of selection pressure: whereas strains 8

from circuit A or D, submitted to a low frequency of heat shocks (circuit A) or to 9

chlorine treatment due to the material of the pipes (circuit D), were shown to remain 10

susceptible to superheating through time, strains from circuit C submitted to an 11

intensive program of heating became rapidly heat-resistant; circuit B offers an 12

intermediate situation since the same clone, initially highly susceptible to heating, 13

became heat-resistant after several years of intensive heat shock procedures 14

(Figures 1 and 4). Genotypic changes similar to those invoked by Chang et al. (9) 15

may explain this spontaneous evolution. Additional experiments are in progress in 16

our laboratory to document the in vitro acquisition of heat resistance by Legionella 17

strains submitted to various stresses, including heat shock, chlorine treatment or 18

starvation in sterile tap water. Further studies should also be undertaken to elucidate 19

the molecular mechanisms involved in the passage from heat-susceptibility to heat-20

resistance; actually; genetic analyses comparing B1 and B2 strains could help to 21

understand the phenotypic changes mentioned above. 22

23

From the practical point of view, this study indicates that FCA could be useful to 24

rapidly evaluate the heat-susceptibility of Legionella strains in order to optimize the 25


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measures used to control their proliferation. Actually, the determination of the 1

proportion of VC and VBNC by FCA after a 30 min heating at 70°C (Figure 4) could 2

help to predict the effectiveness of thermal treatment on a water circuit contaminated 3

by Legionella. Many years before the set-up of a chlorine pump, this measure could 4

have been determinant for deciding for which circuits this preventive measure was 5

necessary and the pursuit of repetitive heat shocks useless. Before FCA may be 6

recommended as an additional tool for the monitoring of the contamination of water 7

circuits by Legionella, these observational data need to be confirmed on a larger 8

number of environmental situations. 9

10

11

ACKNOWLEDGMENTS 12

13

This project was supported financially in part by the Agence Française de Sécurité 14

Sanitaire de l’Environnement et du Travail (AFSSET) and by Microbiodetection SARL 15

(Commercy, France). The authors are indebted to the following contributors of the 16

University hospital of Saint-Etienne: the medical and nursing staff of the Infection 17

control unit for the collection of samples and the monitoring of water circuit treatment, 18

the engineers –and notably François CHORD- who conducted the control measures 19

to decrease Legionella contamination of the water systems, the technical staff of the 20

Laboratory of Bacteriology-Virology-Hygiene for the isolation and collection of strains, 21

particularly Julie RISSOAN and Horia TUZET who performed the AP-PCR 22

experiments. Thibaut EPALLE and Thomas ROS are also acknowledged for skilful 23

technical assistance. 24

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of some halogen biocides using a microbial biofilm system. Water Res 39:4126-32. 13

37. van der Kooij D., H. R. Veenendaal, and W. J. Scheffer. 2005. Biofilm formation 14

and multiplication of Legionella in a model warm water system with pipes of copper, 15

stainless steel and cross-linked polyethylene. Water Res 39:2789-98. 16

38. Withers, H., S. Swift, and P. Williams. 2001. Quorum sensing as an integral 17

component of gene regulatory networks in Gram-negative bacteria. Curr Opin 18

Microbiol 4:186-93. 19

20


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18

Table 1. Measure of percentages of viable cells (VC and VBNC populations) by flow 1

cytometry assay before and after treatment at 70°C. 2

3

Mean percentage [range] of viable cells

Group of strains* (Legionella species)

Number of strains

Before treatment

After 30 min at 70°C

After 60 min at

70°C

A (L. pneumophila)

11

73.9 [35.5 - 92.3]

11.7 [0.8 - 32.4]

9.1 [0.3 - 21.6]

B1 (L. pneumophila) 5 89.9 [86.3 - 94.1] 12.7 [7.3 - 18.4] 7.7 [1.5 - 20.2]

B2 (L. pneumophila) 7 90.2 [80.1 - 95.0] 70.5 [49.1 - 84.7] 57.5 [40.5 - 67.5]

C (L. pneumophila) 13 84.4 [42.5 - 95.1] 71.7 [41.8 - 88.7] 59.9 [39.4 - 84.8]

D (L. anisa) 3 70.9 [58.8 - 79.1] 4.6 [1.4 - 11.8] 2.4 [0.4 - 5.8]

4

* as defined in Figure 4. 5

6


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LEGENDS OF FIGURES 1

2

Figure 1. Chronology of physical treatments applied to four hospital water circuits 3

chronically-contaminated with Legionella. Rectangles correspond to the different 4

decontamination measures as described. Cylinder figures the initiation of a 5

continuous treatment using a chlorine pump. The Legionella strains used in the study 6

are represented by stars positioned at the time of their isolation. 7

8

Figure 2. Representative AP-PCR profiles of strains of Legionella isolated from four 9

different water circuits over a 18 year period with primers G (panel A) and Eric 2 10

(panel B). Ma: size marker; *: negative control. 11

12

Figure 3. Representative flow cytometry patterns of Legionella strains A, B and C (L. 13

pneumophila sg1) and D (L. anisa) after 3 days of culture on BCYE medium. The 14

number under each panel represents the year of isolation of the corresponding strain. 15

Windows allowing determination of viable (green points), VBNC (blue points) and 16

dead (red points) cells percentages are depicted for strains recovered in 2009, 2008, 17

2002 and 2008 from A, B, C and D circuits respectively. 18

19

Figure 4. Resistance of Legionella strains to a heat shock 30 minutes at 70°C. 20

Letters refer to the three water circuits contaminated by strains of L. pneumophila 21

sg1. Bars correspond to the different strains presented in Figure 1. For water circuit 22

B, two successive heat resistance profiles were observed: B1 for strains isolated 23

from 1992 to 1994 and B2 for strains isolated from 2000 to 2008. 24

25


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Figure 5. Viability of L. pneumophila sg 1 strains obtained by flow cytometry assay 1

after treatment at 70°C for 0, 10, 30 and 60 minutes. Letters refer to the group of 2

strains described in Figure 4. Results are expressed as mean ± SE. 3


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Figure 1

Legionella pneumophila sg 1

A (n=11)

��

1998 2006 2008

1 year

1992 2000 201019961994 2002 2004

B (n=12) �

��


C (n=13) �

��


30 min heat shock at 70°C

Chlorine disinfection

Temperature rise to 65°C for at least one day

� Chlorine pump

� Strain of Legionella analysed by FCA and AP-PCR

D (n=3)

Legionella anisa

� � �

�


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A

B

Figure 2

2000 2002 2010 1992 1994 2000 2008 1999 2001 2005 2005 2007 2008 * MaYear of

isolation

Water

circuitA B C D


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Figure 3

FL

3 c

han

el

(red

flu

ore

scen

ce)

(A) (B) (C)

FL1 chanel (green fluorescence)

(D)

2005

2007

2008

2001

2002

2009

1994

2000

2008

1999

2001

2002


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Figure 4

Compromise/VBNC cellsViable and culturable cells Dead cells

A

0%

20%

40%

60%

80%

100%

10/05/2000 01/12/2000 04/01/2001 01/03/2001 27/09/2001 08/10/2001 12/02/2002 16/12/2002 19/01/2004 09/12/2009 14/01/2010

C

0%

20%

40%

60%

80%

100%

11/03/1999 05/07/1999 31/08/1999 27/09/1999 06/12/1999 20/12/1999 19/03/2001 03/09/2001 17/09/2001 17/10/2001 13/06/2002 31/01/2005 21/02/2005

B

0%

20%

40%

60%

80%

100%

25/11/1992 08/04/1994 09/05/1994 30/06/1994 11/07/1994 03/04/2000 17/04/2000 24/05/2000 19/02/2001 28/03/2002 22/09/2008 20/10/2008

B1 B2


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Figure 5

Time at 70°C (min)

Perc

enta

ge

of cells

of

each

cate

gory

(%)

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

Dead cells

Viable cells

Viable but non culturable cells

A B1 B2 C


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