Cell Biology and Toxicology. 2003; 19: 83^93. # 2003 Kluwer Academic Publishers. Printed in the Netherlands
In£uence of peracetic acid on adhesion/invasion of Salmonella enterica serotype Typhimurium LT2 A. Jolivet-Gougeon1, F. Sauvager1, M. Arturo-Schaan1, M. Bonnaure-Mallet2 and M. Cormier1 Equipe de Microbiologie, UPRES 1254, Faculte¨ des Sciences Pharmaceutiques et Biologiques, 2 Equipe de Biologie Buccale, UPRES 1256, U.F.R. Odontologie, Universite¨ de Rennes I, Rennes, France
1
Received 16 May 2002; accepted 14 October 2002
Keywords: Salmonella enterica Typhimurium LT2, adhesion invasion, HeLa cells, anti-actin antibody, peracetic acid Abstract The in£uence of peracetic acid (PAA) disinfectant on Salmonella enterica serotype Typhimurium LT2 in sewage e¥uent was examined by studying its ability to adhere to and invade HeLa cells in vitro. Although the disinfectant produced a decrease of about 5 log units, the bacteria kept their adhesive and invasive abilities. Scanning microscopic observations of the PAA-treated bacteria revealed that PAA caused a loss of external micro¢laments and an alteration of membrane structure. Nevertheless, electron-microscopic observations showed that PAA-treated bacteria were still able to adhere to and invade HeLa cells despite the fact that the bacteria seemed to have undergone some structural modi¢cations. With confocal microscopy, the use of anti-actin antibody showed that the contact between the bacteria (with or without PAA treatment) and the HeLa cells activated actinopolymerization of the HeLa cell cytoskeleton. Abbreviations: AI, association index; ASW, arti¢cial sea water; cfu, colony-forming unit; FITC, £uorescein isothiocyanate; MIC, minimum inhibitory concentration; PAA, peracetic acid; SEM, scanning electron microscopy; S. Typhimurium LT2, Salmonella enterica serotype Typhimurium LT2; SEW, sewage e¥uent water; TRITC, tetramethyl rhodamine isothiocyanate Introduction Members of the genus Salmonella are Gramnegative enteric bacilli that cause salmonellosis or gastroenteritis (Lee and Boey, 1999). Salmonella Typhimurium has been isolated from healthy carriers as well as from sporadic and outbreak cases of salmonellosis (Matsumoto et al., 2000). Electron-microscopic studies by Takeuchi (1967) and Takeuchi and Sprinz (1967) show that an essential step in salmonel-
losis is the invasion of enterocytes in the brush border via a phagocytosis-like process. The bacteria cross the cells and enter the lamina propria. To do this, Salmonella spp. must ¢rst adhere to and subsequently invade the eukaryotic cells (Menard and Sansonetti, 1996). In industrialized countries, salmonellosis is still a major health hazard linked to intensive poultry farming, the production of egg-containing foods, and an increase in the number of human intestinal carriers of Salmonella. In developing
84 countries, poor hygiene standards and unsalubrious sanitary facilities are the main causes of salmonellosis (Sansonetti, 1992). To prevent the health and ecological hazards associated with inadequate wastewater processing, the ¢nal step in the treatment of domestic sewage should involve a disinfection treatment to inactivate pathogens. Peracetic acid (PAA) has been widely used for many years as an e¡ective disinfectant in hospitals and water treatment plants (Baldry and Fraser, 1988; Baldry and French, 1989a; Bonadonna et al., 1999; Stampi et al., 2001). Studies have indicated that PAA is an e¡ective bactericidal (Alasri et al., 1992; Sagripanti et al., 1997; Thamlikitkul et al., 2001), viricidal (Baldry and French, 1989b; Briefman-Kline and Hull, 1960), fungicidal (Baldry, 1983; Lynwood et al., 1967), and sporicidal agent (Baldry, 1983; Baldry and French, 1989b). Secondary e¥uents are commonly disinfected with 0.5^2 mg/L of peracetic acid (Stampi et al., 2001; Wagner et al., 2002) and this disinfection achieves the target levels of fecal indicators. In contrast, we have demonstrated that PAA has no signi¢cant e¡ect on the plasmid content of Escherichia coli strains (Arturo-Schaan et al., 1996) or the enterotoxigenesis of E. coli H10407 (ETEC) (Jolivet-Gougeon et al., 1996). The e¡ect of PAA disinfection on the pathogenicity of Salmonella spp. in wastewater should thus be ascertained, since McFeters et al. (1986) have found that enteric bacterial pathogens such as Salmonella enterica serotype Typhimurium are less susceptible to injury than coliforms. Although the exact mechanism of action is unknown, PAA probably disrupts enzymatic sulfydryl (^SH) and sulfur (S^S) bonds, causing oxidative disruption of major membrane components (Briefman-Kline and Hull, 1960). These alterations may a¡ect the interaction between the bacteria and the eukaryotic cells. The aim of the present study was to determine whether the use of this disinfectant in wastewater can signi¢cantly modify the adhe-
sion/invasion ability of Salmonella enterica serotype Typhimurium. Materials and methods Sample collection Samples of sewage e¥uent water (SEW) obtained from a pilot-scale sewage treatment plant at Saint Pol de Le¨on (Brittany, France) were ¢ltered once through 3 mm pore ¢lters (Millipore) and twice through 0.22 mm pore ¢lters, and then autoclaved for 20 min at 1208C. Samples of arti¢cial sea water (ASW) (Instant Ocean) (33 g/L) were also ¢ltered once through 0.22 mm pore ¢lters and autoclaved as described above. Bacterial strains Salmonella enterica serotype Typhimurium LT2 (S. Typhimurium LT2) was kindly supplied by M. Popo¡ (Institut Pasteur, Paris). E. coli HB101 (ATCC 33694) was used as a negative control in the invasion assay. Peracetic acid treatment PAA was obtained from Seppic Corporation as a 14% aqueous solution (w/v) (Bactipal D1). Water samples were treated for 1 h with 5 mg/L of PAA, corresponding to the MIC of PAA for S. Typhimurium LT2, considered as the optimal cost-e¡ective concentration. The PAA was then inactivated using a 2 g/L solution of sodium thiosulfate. Inoculation of sterilized sewage e¥uent and arti¢cial seawater The bacteria were grown in brain^heart infusion broth (BHI, AES, France) at 378C with shaking until they reached the stationary phase
85 (OD600 nm = 1.2). The bacteria were pelleted, washed once with sterilized SEW, resuspended in sterilized SEW at a concentration of approximately 108 cfu/ml, and incubated for 24 h at 15+18C with shaking. PAA was added to half of the suspension at a concentration of 5 mg/L for 1 h. After inactivation with sodium thiosulfate, the suspensions were diluted 1:5 in sterilized SEW and ASW. The other half, without PAA treatment (control £asks), were concomitantly diluted in sterilized SEW and ASW. The four suspensions were incubated at 15+18C with shaking and samples were removed after 1, 24, and 48 h for the adhesion and invasion assays. The samples were inoculated on MÏller Hinton (MH) agar plates (AES, France) to determine the number of culturable bacteria. HeLa cells The HeLa cells were maintained in minimal essential medium (MEM) (Gibco Laboratories, Grand Island, NY) containing 10% (v/ v) fetal bovine serum and 0.002 mol/L l glutamine (Gibco) at 378C in a 5% CO2^95% air atmosphere. Penicillin^streptomycin (Gibco) was added to the MEM at 100 U/ml and 100 m/ml respectively. The cells were seeded at a concentration of 46105/ml in culture medium in 6-well plates (Nunc, USA) for transmission electron microscopy (TEM), in 24-well plates for invasion assays, in 96-well plates for scanning electron microscopy (SEM), and in 8-well tissue culture slides (Nunc) for the adhesion assays and confocal microscopy. Adhesion assay The assays were performed in 8-well tissue culture slides with 24 h HeLa cell monolayers. Two hours before the assay, the monolayers were washed three times with MEM without antibiotic or serum.
After each sampling procedure, the S. Typhimurium LT2 and E. coli HB101 cells were pelleted, washed twice with washing medium (MEM without antibiotic and serum), and resuspended in washing medium, to give a total bacterial count of approximately 107 cfu/ ml. The bacterial suspensions (0.4 ml) were inoculated into duplicate wells containing HeLa cell monolayers and incubated for 1 h at 378C in a 5% CO2 incubator to allow adhesion. A ratio of bacteria to HeLa cells 4102 was used as recommended by Kusters et al. (1993). The inoculated HeLa cells were then washed ¢ve times with PBS. The monolayers were ¢xed in 95% ethanol for 5 min and stained with Giemsa (diluted 1:10 in water) (Bruchet Labo, France) for 20 min. Bacterial adhesion was assessed by light microscopy at a magni¢cation of 1000. One hundred HeLa cells were examined and the number of adhering S. Typhimurium was counted. The counting procedure was performed in duplicate. The association index (AI) was expressed as the average number of bacteria attached to one HeLa cell. Bacteria from samples with an AI42 were considered associative (Fauche©re et al., 1986). This experiment was carried out four times. Invasion assay The HeLa cells were treated as for the adhesion assay, except that after the PBS wash they were reincubated for 2 h at 378C in a 5% CO2 atmosphere in MEM containing 100 mg of gentamicin/ml (Gibco) but no fetal bovine serum. The monolayers were then washed ¢ve times with PBS and the cells were lysed in 1 ml of distilled water on crushed ice for 15 minutes. The lysates were spread on MH agar plates to determine the number of bacteria that had invaded the HeLa cells (Michelet et al., 1994). Data represent the means of four di¡erent experiments.
86 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) For SEM observations, after a 1 h (adhesion assay) or 3 h (invasion assay) incubation under the conditions described above, the monolayers were washed ¢ve times with PBS, ¢xed with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate^HCl bu¡er (pH 7.2) for 1 h at room temperature, and rinsed in the same bu¡er. The cells were dehydrated with increasing concentrations of ethanol (50% to 100%), then placed in a Balzers CPD 010 for critical point drying. The specimens were coated with a thin ¢lm of gold^palladium (60/40) using a JEOL JFC 1100 and examined using a JEOL JSM-6400 scanning electron microscope at 11 kV. For TEM observations, after a 1 h or 3 h incubation under the conditions described above, infected cells were post¢xed in 1% osmium tetroxide followed by dehydration. They were then embedded in Epon-Araldite. Ultra-thin TEM sections (800 Aî) were deposited on copper grids (200 mesh), contrasted with uranyl acetate and lead citrate, and examined using a JEOL SEM 100CXII at 80 kV. For determination whether the PAA treatment had a¡ected the bacterial surface, untreated and PAA-treated bacteria without a HeLa support were observed by SEM. Samples were centrifuged and the pellets were resuspended in PBS. Drops of resuspended bacteria were then placed in 96-well plates and treated as described above. Triplicate wells were prepared for each sample. Confocal microscopy The procedure was essentially the same as for the adhesion assay, except that the monolayers were incubated with a 1:8 dilution of mouse serogroup B-speci¢c monoclonal antibody (provided by Dr. Desmonts, UPRES 1254, Rennes, France) for 30 min. After washing
with PBS, the monolayers were treated for 30 min with a 1:100 dilution of anti-mouse IgG tetramethyl rhodamine isoth iocyanate (TRITC) conjugate (Sigma). The monolayers were then washed and treated with a 1:50 dilution of monoclonal anti-actin £uorescein isothiocyanate (FITC) conjugate (Sigma) for 30 min. The slides were washed, a coverslip was mounted on each slide with a drop of 30% glycerol in PBS pH 7.4, and the edges were sealed with nail polish. The slides were examined using a confocal laser scanning microscope (Leica TCS NT) at 590 nm for TRITC and 530 nm for FITC. PAA-treated and untreated Salmonella cells were stained with a 1:4 dilution of rabbit anti£agella polyclonal antibody (Biorad) for 30 min. After washing with PBS, bacteria were labeled with a 1:100 dilution of anti-rabbit IgG £uorescein isothiocyanate (FITC) conjugate. The slides were observed as described above. Results Bacterial counts When S. Typhimurium LT2 cells were incubated for 24 h in SEW, the plate counts remained constant, typically 108 cfu/ml. The treatment of SEW with 5 mg/L of PAA for 1 h produced a sharp drop in Salmonella numbers (5 log units below the initial level, or approximately 103 cfu/ml). After inactivation with sodium thiosulfate and dilution in SEW or ASW, the plate counts increased slowly and reached 106 cfu/ml, with no signi¢cant di¡erence between regrowth in SEW or ASW after 48 h (data not shown). Small variations in numbers were observed, depending essentially on the di¡erent SEW samples.
87 Evaluation of the adhesive and invasive abilities of Salmonella typhimurium by Giemsa staining of con£uent monolayers of HeLa cells and enumeration of intracellular bacteria Microscopic examinations of Giemsa-stained con£uent monolayers of HeLa cells incubated with PAA-treated Salmonella Typhimurium after dilution showed that the bacteria in the SEW and ASW samples were considered as not associative (AI52) 1 and 24 h post disinfection (Table 1), whereas they retained their invasive ability (the number of intracellular bacteria was about 102 cfu/ml) (Table 2). After 48 h in SEW or ASW, PAA-treated Salmonella had AI = 2.4+0.9 and 10+3.0, respectively (Table 1), and the numbers of intracellular bacteria were (1.1+0.4)6103 and (4.8+3.9)6104/ml, respectively (Table 2), indicating that their ability to invade HeLa cells was preserved. The AIs of the other samples (without PAA treatment) indicated that the Salmonella Typhimurium cells remained associative (AI 42) (Table 1), while the number of bacteria recovered from the lysed HeLa cells ranged from 103 to 105 cfu/ ml (Table 2).
Scanning electron microscopy Adhesion studies. We observed that control Salmonella cells adhered to HeLa cells, produced many external micro¢laments, and started to internalize into the HeLa cells (Figure 1a). The SEM examinations of HeLa cells incubated with PAA-treated-bacteria showed that these bacteria also adhered to the cells (Figure 1b), although they had a low AI, were less numerous on the HeLa cell monolayers than untreated bacteria, and did not produce the long ¢laments observed in the control assay. Invasion studies. Untreated Salmonella caused considerable ru¥ing of HeLa cell membranes. In contrast, PAA-treated bacteria bound to the HeLa cells, but did not initiate the internalization process and had only a few ¢laments on their surfaces. However, the treated bacteria caused considerable disorganization and blebbing of the HeLa cells. Direct impact of PAA on bacteria in the absence of HeLa cells. SEM examinations of bacteria in the absence of HeLa cells revealed that PAAuntreated bacteria (Figure 2a) had conserved most of their cell surface micro¢laments, while the treated bacteria had not (Figure 2b).
Table 1. Microscopic e6aminations of Giemsa-stained con£uent monolayers of HeLa cells and association index (AI) measurements (Fauche©re et al., 1986) to study adhesive capacities of Salmonella Typhimurium LT2. The bacteria were pelleted in stationary phase culture and resuspended in sewage e¥uent water (SEW). Half of the microcosm was treated with 5 mg/L of PAA for 1 h, then diluted 1:5 in sterilized SEW or arti¢cial sea water (ASW). Samples were removed after 1, 24, and 48 h post disinfection for the adhesion assay. Association index (AI) With PAA treatment Time postdisinfection 1h 24 h 48 h
Without PAA treatment
SEW
ASW
SEW
ASW
0.6+0.5 1.0+ 1.0 2.4+ 0.9
1.1+ 0.9 1.7+ 0.1 10.0+ 3.0
5.5+ 0.1 19.4+ 0.4 16.0+1.0
6.1+ 0.1 8.0+ 1.8 9.0+ 0.5
88 Table 2. Invasion of con£uent monolayers of HeLa cells by Salmonella Typhimurium LT2 was assayed by enumerating intracellular bacteria, with or without PAA treatment of sewage e¥uent water in the same conditions as for AI measurements. No. of intracellular bacteria per millimeter With PAA treatment Time postdisinfection 1h 24 h 48 h
SEW
Without PAA treatment
ASW 2
(0.2+0.4)610 (1.2+1.2)6102 (1.1+0.4)6103
SEW 2
(3 +3)610 (2.8+1.9)6102 (4.8+3.9)6104
ASW 3
(7 +3.2)610 (7.8+3.1)6103 (3.2+2)6103
(1.5+1.2)6103 (1.2+0.7)6105 (1.8+0.2)6104
a
a
b b Figure 1. Scanning electron micrographs of HeLa cells incubated with Salmonella Typhimurium LT2 for 1 h. Incubation of HeLa cell monolayers with (a) untreated Salmonella Typhimurium (positive control) and (b) PAAtreated bacteria. Bars represent 1 mm.
Figure 2. Scanning electron micrographs of Salmonella Typhimurium LT2 (a) before and (b) after exposure for 1 h to 5 mg/L of PAA. Bars represent 1 mm.
89 Transmission electron microscopy Adhesion studies. Observations of HeLa cells after a 1 h incubation with untreated or PAAtreated Salmonella revealed connections between untreated bacteria and HeLa cells after 1 h of contact. Internalized bacteria were observed in intracytoplasmic vacuoles. The adhesion and internalization processes of PAA-treated bacteria did not change, although compared with untreated bacteria some alterations in their outer membranes were observed (data not shown). Invasion studies. A transverse section of internalized treated and untreated bacterial cells after 3 h of contact indicated that the PAAtreated Salmonella also retained their invasive ability (data not shown). Indirect £uorescent-antibody staining. Indirect £uorescent-antibody staining revealed that actin polymerization, which is part of the invasion process, occurred in the HeLa cell monolayers. The bacteria stained red with the TRITC conjugate (Figure 3a), and actin accumulation was observed (FITC conjugate) on the same slide, as a uniform green capsule surrounding each bacterial cell (Figure 3b). No £uorescence was observed with the negative control (E. coli HB101). Salmonella without HeLa cells and HeLa cells without Salmonella did not £uoresce following anti-actin antibody staining. Moreover, the PAA treatment of the bacteria did not a¡ect actin polymerization. Anti£agellin antibody staining showed that £uorescence appeared around treated as well as untreated bacteria, but a comparison of the levels of £uorescence was di¤cult (Figure 4).
a
b Figure 3. Fluorescent micrographs of HeLa cells 1 h after incubation with Salmonella Typhimurium LT2. The HeLa cells were incubated with mouse serogroup B-speci¢c monoclonal antibody, then with anti-mouse IgG TRITC conjugate, and lastly with monoclonal anti-actin FITC conjugate. The di¡erent structures were observed using the following wavelengths. (a) 590 nm: on HeLa cell monolayers, visualization of Salmonella Typhimurium at the same spot at which the actin condensation is shown in (b). (b) 530 nm: visualization, on the same HeLa cell monolayers, of anti-actin antibody showing concentrated areas of bacteria-associated £uorescence.
90
Figure 4. Fluorescent micrograph of anti-£agella antibody staining labeled with an anti-rabbit IgG £uorescein isothiocyanate conjugate, showing some broken £agella and others linked to PAA-treated bacteria.
Discussion The disinfection e¤cacy of PAA was evaluated using Salmonella Typhimurium, which is a major cause of diarrheal disease as well as a common indicator of fecal contamination. For these experiments, Salmonella Typhimurium was grown to the stationary phase before adding the PAA in order to obtain conditions similar to those of a natural environment in terms of temperature, e¥uent dilution in sewage treatment plants, and dilution in sea water (Gauthier et al., 1993). However, the bacterial inoculum is a limiting factor and has to be adjusted to 107^108 bacteria per ml (a higher level than those encountered in secondary e¥uent water), to ensure accurate AI values (Kusters et al., 1993). In common conditions, disinfection with PAA in sewage treatment plants reduce levels of fecal contamination by 97% (Stampi et al., 2001) and PAA is therefore
considered as e¡ective in secondary e¥uents with a high degree of puri¢cation. PAA activity can vary with the dose and contact time of the disinfectant, and with environmental conditions such as bacterial competition or amount of dissolved organic matter in natural waters (Braux, 1998; Bonadonna et al., 1999). The drop in the number of bacteria varies with the bacterial group studied, because the MIC of PAA depends on the strain (more e¡ective against coliforms than Pseudomonas spp. or staphylococci) (Bonadonna et al., 1999). As has been shown for E.coli under both real-life (Arturo-Schaan et al., 1996) and laboratory (Jolivet-Gougeon et al., 1996) conditions, the treatment of e¥uents with 5 mg/L of PAA causes a major reduction in Salmonella Typhimurium numbers. Some authors (Ernst et al., 1990; Lee and Falkow, 1990) have reported that growth conditions and growth phase may a¡ect the ability of Salmonella to invade cultured cells. Anaerobically grown and log-phase cultures are more invasive than aerobically grown and stationary phase cultures. However, one study has demonstrated that the ability of Salmonella to adhere to and invade cultured cells is independent of the bacterial growth phase, but is related to the ratio of HeLa cells to bacteria, called ``mutiplicity of infection'' by Kusters et al. (1993). HeLa cells were used because they are easily cultured and provide a good model for studying bacterial adhesion and invasion (Fauche©re et al., 1986; Park et al., 2000). The reproducibility of the AI has been veri¢ed and Fauche©re et al. (1986) have shown that it is a reliable, convenient, and rapid method for estimating the pathogenicity of Campylobacter strains. The number of culturable bacteria recovered 48 h after PAA treatment reached 106 cfu/ml. Both 1 h and 24 h after PAA treatment, injured Salmonella were not considered as associative; but after 48 h in ASW, PAA-treated Salmonella seemed to recover their adhesive capacities, reaching an AI42. After 48 h, the recovery of
91 adhesive capacities in SEW is more uncertain, as attested by statistical analysis, but is con¢rmed after 72 h (data not shown). This recovery therefore seemed to be faster in ASW than in SEW, and this could be explained by dissolved substances in SEW able to inhibit interactions with HeLa cells (Stenstrom and Kjelleberg, 1985). On the other hand, an oxidative stress could allow bacterial adaptation to osmotic stress and enhance faster metabolic recovery (Braux, 1998; Rozen and Belkin, 2001), but these results greatly depended on the SEW sample used. However, if only a few bacteria did adhere to the HeLa cells, they were able to invade them and multiply, which led (after the 3 h incubation time) to a number of intracellular bacteria per ml considered as signi¢cant. Several authors have also reported that peracetic acid can cause mutations (Koch et al., 1989). Peracetic treatment could be a condition in which Salmonella undergo rapid evolution by natural selection and mutants displace their wild-type parents as the majority. This remains possible despite the fact that no phenotypic variations were detected among the PAA-treated bacteria that survived. In response to adverse environmental conditions, bacteria can enter the viable but nonculturable state, and conserve their adhesive capacities (Rahman et al., 1996). The regrowth of treated bacteria could also be due to resuscitation of viable nonculturable bacteria. Survival of enteric pathogens exposed to various environmental stresses depends upon a number of protective responses, some of which are associated with induction of virulence determinants. Many ¢laments were observed by SEM on the surfaces of the bacteria after 1 h of contact with the HeLa cells. Some authors have shown that Salmonella Typhimurium type 1 ¢mbriae mediate adhesion to HeLa cells in vitro (Ernst et al., 1990; BaÏmler et al., 1996). Flagella may also play an important role in the process by which
bacteria adhere to and invade cells (Darwin and Miller, 1999; Schmitt et al., 2001). Walsh and Bissonnette (1983) have demonstrated that chlorine-injured E. coli cells in fresh water lose their £agella. From the SEM results reported here, it can be assumed that PAA-treated bacteria without HeLa support rapidly lost only some of their ¢laments, but interaction with HeLa cells seemed to protect some of them from PAA injury. These ¢lamentous structures were not £agella, as attested by positive £uorescent anti-£agella staining in PAA-treated and non-treated bacteria, and could be ¢mbriae or protein aggregates. Following an additional 2 h of incubation for invasion assay, the PAA-treated bacteria started to partially re-synthesize them and also produced a cell reaction resulting from an environmental aggression. Positive £uorescence could also be due to scarce surviving bacteria that had multiplied during 2 h of invasion-incubation time. It has been demonstrated with Shigella £exneri (Sansonetti et al., 1994), Salmonella Typhimurium (Menard and Sansonetti, 1996), and Listeria (Mengaud et al., 1996) that internalization of bacteria results in a reorganization of the cytoskeleton of the eukaryotic cell (Francis et al., 1992; Francis et al., 1993; Gala©n, 1999). In the work presented here, we also con¢rmed that actin was involved in the Salmonella Typhimurium invasion process; PAA treatment of bacteria before HeLa cell contact produced no visible alterations of this phenomenon as observed by confocal laser scanning microscopy. In conclusion, PAA treatment (5 mg/L) of wastewater had no apparent e¡ect on the virulence of Salmonella Typhimurium since the bacteria retained their ability to adhere to and invade HeLa cells. This indicates that there may a potential risk of pathogenic bacteria disseminating in natural and bathing water.
92 Acknowledgments This work was supported by the European Community (contract Newtech: EV5V-CT940400). We thank Joe«lle Mounier (Institut Pasteur, Paris) for providing the HeLa cells and Michel Popo¡ (Institut Pasteur, Paris) for providing the Salmonella enterica Typhimurium LT2. We also thank R. Primault and J. Le Lannic for their assistance with the transmission and scanning electron microscopy, respectively, S. Tricot-Doleux for her technical assistance, and J. Gue¨rin for statistical analysis. The authors are grateful to G. Bourgeau and G. Boue«r for revising the English translation. References Alasri A, Roques C, Michel G, Cabassud C, Aptel P. Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains. Can J Microbiol. 1992;38: 635^42. Arturo-Schaan M, Sauvager F, Mamez C, Gougeon A, Cormier M. Use of peracetic acid as a disinfectant in a watertreatment plant: e¡ect on the plasmid contents of E. coli strains. Curr Microbiol. 1996;32:43^7. Baldry MGC. The bactericidal, fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid. J Appl Bacteriol. 1983;54:417^23. Baldry MGC, Fraser JAL. Disinfection with peroxigens. Industrial biocides. Critical reports and applied chemistry. 1988;23:91^116. Baldry MGC, French MS. Disinfection of sewage e¥uent with peracetic acid. Water Sci Technol. 1989a;21:203^6. Baldry MGC, French MS. Activity of peracetic acid against sewage indicator organisms. Water Sci Technol. 1989b;21: 1747^9. Ba«umler AJ, Tsolis RM, He¡ron F. Contribution of ¢mbrial operons to attachment to and invasion of epithelial cell lines by Salmonella Typhimurium. Infect Immun. 1996;64:1862^ 5. Bonadonna L, Della Libera S, Veschetti E, et al. Reduction of microorganisms in sewage e¥uent using hypochlorite and peracetic acid as disinfectants. Cent Eur J Public Health. 1999;7:130^2. Braux AS. Pro¢ls prote¨iques et adaptation de Escherichia coli en e¥uent de station d'e¨puration et apre©s de¨sinfection a© l'acide perace¨tique. [Doctoral thesis], Universite¨ de Rennes 1, France; 1998.
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