- Email: [email protected]
Passage in mice causes a change in the ability of Salmonella enterica serovar Oranienburg to survive NaCl osmotic stress: resuscitation from the viable but non-culturable state
FEMS Microbiology Letters 212 (2002) 87^93
www.fems-microbiology.org
Passage in mice causes a change in the ability of Salmonella enterica serovar Oranienburg to survive NaCl osmotic stress: resuscitation from the viable but non-culturable state Hiroshi Asakura a , Sou-ichi Makino a; , Tsuyoshi Takagi a , Asumi Kuri a , Takayuki Kurazono b , Masahisa Watarai a , Toshikazu Shirahata a a
Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555 Japan b Saitama Prefectural Institute of Public Health, Saitama 338-0824 Japan Received 6 February 2002; received in revised form 24 April 2002 ; accepted 26 April 2002 First published online 29 May 2002
Abstract An outbreak caused by dried processed squids contaminated with Salmonella Oranienburg occurred in Japan in 1999. Isolates obtained from the causative food were resistant to NaCl osmotic stress, but isolates from the patients were sensitive to NaCl. Although strains from both sources were almost identical in their virulence in mice, a NaCl-resistant strain from food (Sa9911T) became NaCl-sensitive after passage in mice and a NaCl-sensitive strain from one patient (Sa99004) retained NaCl sensitivity after such passage. When dried squid was contaminated experimentally with both strains during processing, only Sa9911T was recovered directly from the final product. Nevertheless, the viability of the Sa99004 cells was over 90% found by fluorescent staining. We suggested that Sa99004 might become viable but non-culturable (VNC) by NaCl stress. This hypothesis was confirmed by resuscitation by efficient enrichment. We concluded that VNC S. Oranienburg would be potentially dangerous contaminants of NaCl-preserved foods and that measures to ensure their detection should be taken at the time of food inspection. 8 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Salmonella Oranienburg; Dried squid; NaCl-resistance ; Viable but non-culturable
1. Introduction The preservation of foods by concentrated NaCl followed by desiccation has been used widely as an e¡ective means of preventing food borne infections [1^5]. However, a di¡use outbreak caused by dried squid, which was contaminated with Salmonella enterica subsp. enterica serovar Oranienburg, occurred in 46 prefectures in Japan in 1999. In the outbreak, over 1500 people su¡ered septicemia and gastroenteritis [1,6]. As all the causative foods were manufactured by the same company, the raw squid was probably contaminated with S. Oranienburg during the production process. This led us to hypothesize that S. Oranienburg can survive and retain its pathogenicity for humans in concentrated NaCl and during desiccation.
* Corresponding author. Tel. : +81 (155) 49 5386; Fax : +81 (155) 49 5386. E-mail address : [email protected] (S.-i. Makino).
Several bacterial pathogens, such as Campylobacter jejuni, Listeria monocytogenes, Escherichia coli and Bacillus cereus, have been detected in dried foods [2,7^12]. In addition, Vibrio cholerae, E. coli O157:H7, C. jejuni and Salmonella spp. become viable but non-culturable (VNC) in environments such as seawater, ground water, rivers and salted food [13^19]. This may result in an underestimation of the true number of viable cells in foodstu¡. In this study, we attempted to clarify how and why S. Oranienburg cells survive under harsh conditions. We showed that the isolates from food remained viable after soaking in NaCl and during dehydration, whereas the isolates from patients did not. Sensitivity to NaCl stress was con¢rmed as the phenotype responsible for the di¡erence in survival rates between these isolates, rather than low water activity or temperature. The importance of the bacterial VNC state during manufacturing processes, the resuscitation of VNC cells, and the potential risk they pose to humans are discussed.
0378-1097 / 02 / $22.00 8 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 1 1 - 5
FEMSLE 10508 12-6-02
88
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
2.4. Survival of S. Oranienburg strains in a NaCl solution
2. Materials and methods 2.1. Salmonella strains and media Nine strains of S. Oranienburg associated with the outbreak and two strains originating from sporadic cases were used in this study (Table 1). The estimated water activity of the dried squid was 0.6 [1]. The liquid culture media included nutrient broth (NB; Difco) lacking NaCl, Hajna tetrathionate broth (HTB ; Eiken, Japan), and brain heart infusion broth (BHI; Difco). Agar plates were made from desoxycholate hydrogen sul¢de lactose agar (DHL; Eiken) and trypticase soy agar (TSA ; BBL/Becton Dickinson). 2.2. Analysis by pulsed ¢eld gel electrophoresis (PFGE) Fingerprinting of bacterial DNA by PFGE was done mainly by the method of Gautom et al. [20] with the following modi¢cations. Each chromosomal DNA sample was digested with 30 U of XbaI or BlnI (TOYOBO) and the resultant fragments were analyzed by electrophoresis through a 1% agarose gel (Amersham) in 0.5UTris^borate^EDTA bu¡er, using Gene Navigator (Amersham). 2.3. Sensitivity to drying Bacterial cells grown at 37‡C for 20 h in NB were washed once with sterile saline, then were resuspended in an identical volume of sterile saline. A 50-Wl aliquot of the suspension was mixed with 450 Wl of sterile saline in each well of a 48-well microtiter plate (Nalge Nunc). The plate was incubated uncovered at 45‡C for 24 h to dry the suspension. Sterile water (500 Wl) was then added to each well, and 0.1 ml of the resultant suspension was spread on DHL and TSA plates after serial dilution to measure the colony forming units (CFU).
Table 1 S. Oranienburg strains used in this study Strains
Origin
Symptoms
Sa99001 Sa99004 Sa99026 Sa99061 Sa99073 Sa9911T Sa9912A Sa9921K Sa9937O Sai-1 Sai-2
Patient patient patient patient patient dried squid raw materials raw materials raw materials patient patient
asymptomatic septicemia septicemia septicemia asymptomatic ^ ^ ^ ^ septicemia diarrhea
Approximately 108 ^109 S. Oranienburg cells grown at 37‡C for 20 h in NB were suspended in 10 ml of NaCl solution at various concentrations and were incubated at 37‡C for 24 h. A 0.1-ml aliquot of the suspension and its serial dilutions were spread on DHL and TSA plates to measure the CFU. To ensure the data were accurate, the experiment was repeated in triplicate. 2.5. Experimental infection of mice with S. Oranienburg A total of 50 BALB/c female mice aged 6 weeks were injected intraperitoneally with 6.6U105 S. Oranienburg cells grown at 37‡C for 18 h in NB. At 1 day after infection and then weekly for up to 4 weeks, ¢ve mice were killed with chloroform to collect their liver, spleen, blood, and intestinal contents. Tissue samples were weighed and homogenized in 1 ml PBS. Aliquots of the suspensions were spread on DHL plates to measure the CFU. 2.6. Recovery of Salmonella cells from experimentally infected dried squid 50 ml of 7% NaCl solution containing 5 g of raw squid was inoculated with 6.6U109 S. Oranienburg cells were grown at 37‡C for 18 h in NB, and were incubated at 4‡C for 24 h. The squid was removed, dried at 45‡C for 24 h, soaked in 5 ml bu¡ered peptone water (BPW, pH 7.2), and incubated at 37‡C for 1 h. Aliquots (0.1 ml) of serially diluted samples were spread on DHL plates to measure the CFU. At the same time, 1 ml of the suspension and its serial dilutions were inoculated in 10 ml of HTB, and were incubated at 37‡C for 20 h to isolate Salmonella cells and to calculate the most probable number (MPN) of these cells. Another enrichment procedure using ¢lter-sterilized NB culture supernatant from an overnight culture of strain Sa9911T or Sa99004 was used, essentially by the method of Santo Domingo et al. [19]. 1 ml of the BPW suspension containing squid and its serial dilutions were added to 10 ml of 5% BHI containing 100 Wl of the supernatant, followed by incubation for 7 days at room temperature. To examine the e¡ect of the supernatant on enrichment, 5% BHI without the supernatant was used as a control. Aliquots of the cultures were spread on DHL plates to isolate Salmonella cells and to measure the MPN. 2.7. Determination of viability
Nine strains except Sai-1 and Sai-2 were associated with an outbreak caused by dried squid. The other two strains were isolated from sporadic outbreaks.
Salmonella cells in BPW suspension with squid and in NaCl solutions were stained by using the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes). The bacteria were examined under £uorescence microscopy and the viability of 1000 cells was assessed.
FEMSLE 10508 12-6-02
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
89
3.2. Sensitivity of S. Oranienburg strains to drying or NaCl stress
Fig. 1. PFGE patterns of S. Oranienburg strains. Five representative strains containing two S. Oranienburg strains, Sai-1 and Sai-2, isolated from other sporadic outbreaks, were analyzed by PFGE using BlnI (lanes 1^5) and XbaI (lanes 6^10). Lanes 1 and 6, Sai-1; 2 and 7, Sai-2; 3 and 8, Sa99004; 4 and 9, Sa9911T; 5 and 10; Sa9921K.
3. Results 3.1. DNA analysis of S. Oranienburg strains Epidemiological studies [1,6] have suggested that S. Oranienburg isolates from patients in the outbreak were the same as isolates from the causative food, dried squid. To con¢rm the relationship, nine isolates listed in Table 1 were examined genetically. As expected, their PFGE patterns were identical (Fig. 1, lanes 3^5 and 8^10), but differed from two strains from unrelated sporadic cases (Fig. 1, lanes 1, 2, 6 and 7).
Nine isolates from the outbreak were suspended in saline to ¢nd their sensitivity to drying. First, the suspensions were maintained at 45‡C for 24 h, but no marked di¡erence was observed in survival rates between isolates from food and from patients (Table 2). After drying in saline, however, the CFU of ¢ve strains from patients decreased markedly, especially strain Sa99004 formed no colonies after drying, but four strains from food survived in large numbers and Sa9911T was the most resistant to drying (Table 2), resulting in strains from patients being sensitive to drying in NaCl while those from food were resistant. However, when their sensitivity to drying was examined in sterile water instead of saline, all the isolates appeared to be resistant (Table 2). Therefore, we concluded that sensitivity to drying was concurrent with sensitivity to NaCl. The changes in CFU of each strain in various NaCl concentrations was examined. With increasing NaCl concentrations, the number of the remaining four strains from patients declined more slowly than Sa99004. However, the decline was greater than that of the strains from food (Fig. 2). Thus, strains from food were resistant to NaCl stress, but those from patients were sensitive. The NaCl sensitivity-resistance phenotype of these strains remained stable, even though they were subcultured in NB for 100 passages (data not shown). The CFU of strains from food decreased by approximately 100-fold after incubation for 24 h in 17% NaCl, whereas strain Sa99004 of patient origin formed no colonies after 24 h in 7% NaCl (Fig. 2). Almost identical results were obtained by using DHL and TSA plates for the growth of S. Oranienburg cells after they were incubated in NaCl solution (data not shown). 3.3. Pathogenicity of S. Oranienburg strains in mice As the NaCl sensitivity of S. Oranienburg isolates from
Table 2 Survival of S. Oranienburg isolates through the drying process Strains
Patient origin Sa99001 Sa99004 Sa99026 Sa99061 Sa99073 Food origin Sa9911T Sa9912A Sa9921K Sa9937O
Culturable cell numbers (CFU ml31 ) Initial dose
Drying in saline
Drying in water
Incubation in salinea
1.7U109 5.3U108 4.4U108 6.8U108 3.8U108
3.5U102 6 10 1.9U102 2.4U102 1.5U102
6.9U106 1.8U107 2.2U107 1.4U106 2.5U107
2.6U107 2.2U107 1.7U107 2.8U107 1.2U107
5.6U108 3.7U108 5.0U108 9.4U108
2.8U107 8.9U105 2.3U106 7.2U106
3.0U106 1.0U107 5.6U106 1.4U107
1.8U107 2.5U107 2.2U107 2.7U107
a
All isolates were incubated in saline at 45‡C for 24 h. Each value shows the standard values in the experiments done in triplicate.
FEMSLE 10508 12-6-02
90
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
Fig. 2. Bacterial cell numbers of S. Oranienburg in NaCl solution. The values are the standard means of three experiments.
food and patients were di¡erent, the fact that their pathogenicity might also di¡er was plausible. This possibility was examined in mice by intraperitoneal injection of 6.6U105 CFU of strains Sa9911T and Sa99004, which were selected as the representative isolates from food and patient, respectively. For at least 40 days, all mice survived with no symptoms. In the CFU of bacteria recovered from liver, spleen, blood and intestinal contents, there was the two groups of mice showed no signi¢cant di¡erence for up to 4 weeks after injection (Fig. 3). In this experiment, no signi¢cant di¡erence was detected in pathogenicity for mice. The pathogenicity of NaCl-stressed isolates was compared; NaCl-stressed Sa9911T cells were recovered from organs at similar numbers (Fig. 3), but NaCl-stressed Sa99004 cells were not recovered. This was consistent with the observation that NaCl-stressed Sa99004 cells did not form any colonies on the plates (Fig. 2). 3.4. Phenotypic change in NaCl sensitivity by passage in animals A total of 50 colonies were selected from mice infected with S. Oranienburg strains, and their sensitivity to NaCl
was examined by testing their resistance to drying. Of the Salmonella colonies recovered from mice infected with the NaCl-resistant strain, 4^88% Sa9911T were sensitive to NaCl during passage in vivo, but all the colonies derived from the NaCl-sensitive strain Sa99004 remained phenotypically unchanged. The NaCl resistance-sensitivity phenotype of the colonies derived from Sa9911T recovered from mice remained stable, even though they were subcultured in NB for 100 passages (data not shown). 3.5. NaCl sensitivity of S. Oranienburg in experimentally processed dried squid When the strains from patients were incubated in 17% NaCl solution, the suspensions contained viable cells (Fig. 2), but they remained sensitive to NaCl (data not shown). However, ¢nding if they become resistant to NaCl after incubation in dried squid was necessary. Strains Sa99004 and Sa9911T (approximately 6.6U109 cells) were incubated separately in 7% NaCl solution containing raw squid at 4‡C for 24 h. The squid was then processed experimentally. Each dried squid was soaked in BPW at 37‡C for 1 h, and then 0.1 ml of the suspension and serial dilutions were spread on DHL plates. Although approximately 4.0U108
FEMSLE 10508 12-6-02
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
91
Fig. 3. Comparison of pathogenicity of S. Oranienburg strains in mice. Strain Sa9911T of food origin (A) and strain Sa99004 of patient origin (B) were intraperitoneally injected into BALB/c mice and the viable bacteria were counted in liver, spleen, peritoneal blood, and feces every week for 4 weeks after infection.
cells were recovered from 1 g of dried squid contaminated with strain Sa9911T; no Sa99004 cells were recovered after the same treatment (Table 3). The BPW suspensions were examined for cell viability by staining with £uorescent dye; over 90% of bacterial cells from food and patients were viable among a total of 1000 bacterial cells selected randomly (data not shown).
richment cultures contained Salmonella cells and no cells were detected by direct plating (Table 3), Sa99004 cells must have entered the VNC state and been resuscitated successfully in both enrichment cultures (Table 3). The sensitivity to NaCl of 50 bacterial colonies, which were recovered from dried squid by enrichment plating, remained stable, even though they were subcultured in NB for 100 passages (data not shown).
3.6. Recovery of VNC Salmonella cells Salmonella cells were recovered by enrichment culture in HTB and the MPN was measured simultaneously. The squid BPW suspension from strain Sa99004 was estimated to contain 0^45 (SD = 17.76) viable cells per ml (Table 3). A second enrichment procedure was done by using the culture supernatant [19]. By this method, the suspension was estimated to contain 225^550 (SD = 187.64) viable cells per ml (Table 3). The culture supernatant of strains Sa9911T and Sa99004 caused resuscitation. As all the en-
4. Discussion A di¡use outbreak caused by dried squid contaminated with S. Oranienburg occurred in Japan in 1999 [1,6]. As the isolates from food and patients showed identical PFGE patterns in this study, we concluded that S. Oranienburg contamination of the food caused the outbreak. Bacterial growth is generally poor in foods with a low water activity of around 0.6 [5], therefore the raw materi-
Table 3 Resuscitation of S. Oranienburg cell suspension from the VNC state Enrichment
Recovery of Sa99004 cells froma
Procedures Direct plating
1 ml
100Wl
10Wl
1Wl
SD
(0/25) + (5/25) + (23/25)
(0/25) + (1/25) + (13/25)
(0/25) 3 (0/25) + (5/25)
(0/25) 3 (0/25) 3 (0/25)
0 0^45 17.76 225^550 187.64
HTB Supernatant
MPN values
a
1 ml and its serial dilutions of the BPW suspensions contaminated with Sa99004 cells with dry-processed squids were used for each experiment as described in Section 2. In a total of 25 tubes, the numbers of Salmonella cells recovered in vitro are shown in parentheses. + and 3 indicate that Salmonella cells can or can not be recovered from the samples, respectively.
FEMSLE 10508 12-6-02
92
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
als of the dried squid appeared to have been highly contaminated with S. Oranienburg before or during processing. Estimation by the MPN method indicated that approximately 10^103 cells of S. Oranienburg were present in each gram of the contaminated food, but only fewer than 20 cells were isolated by direct plating [1]. This level of S. Oranienburg contamination was unlikely to establish septicemia, the main symptom of the patients in this outbreak (Table 1). However, if a large proportion of the bacterial cells in the dried squid had entered the VNC state, a much higher number of viable cells, which are intractable under normal culture methods, could have contaminated the food without being detected and caused the outbreak. Salmonella cells rapidly enter the VNC state in aqueous conditions [14,17,19] and their numbers detected in the dried squid gradually decrease during storage [1]. Thus, precise enumeration of bacteria in food is not easy and predicting if a particular level of contamination results in human infection is extremely di⁄cult. In this study, the sensitivity of the strains from food or patients to drying in saline and in distilled water was tested. Even if the initial NaCl concentration is low, the concentration gradually increases during the drying process. Therefore, the sensitivity of bacteria to drying in saline would re£ect their sensitivity to NaCl. As the resistance and sensitivity to drying was consistent with resistance and sensitivity to NaCl, the tests of sensitivity to drying used in this study would be a simple and useful screening method to predict the NaCl sensitivity of the bacteria. This study showed that strains from food were resistant to NaCl and those from patients were sensitive. The NaClresistant strain from food became sensitive following passage in mice, although the basic mechanism for this observation was not clear. This one-way phenotypic conversion occurred only in vivo, and the NaCl resistance in strains of food origin remained unchanged in vitro. We reported previously an outbreak of food poisoning caused by enterohemorrhagic E. coli (EHEC) O157 in contaminated salted salmon roe [16]. What was observed in this outbreak is consistent with the results of this study. In the outbreak due to contaminated salmon, EHEC O157 isolated from patients were sensitive to NaCl while strains from food were resistant. Furthermore, the NaCl-resistant O157 strain also became NaCl-sensitive after passage in vivo. We suggest that the natural phenotype of enteric pathogens like Salmonella and O157 in the environment is NaCl-resistant. The results of this study showed that the pathogenicity for mice of NaCl-sensitive and -resistant isolates was almost the same, and we conclude that the NaCl sensitivity is not related to the virulence. Under various stressful conditions [13^19,21,22], bacterial cells may become VNC as a protective mechanism. Resuscitation could occur under certain conditions [19,23,24], and hence bacterial cells becoming VNC might
protect against environmental stress. In this study, the resuscitation of Salmonella cells from the VNC state was shown in vitro by two enrichment procedures. These methods might be useful not only to detect Salmonella cells, but also to estimate viable numbers of other bacterial pathogens that have become VNC in foods. The genetic response induced by NaCl osmotic stress has been reported for several bacterial species [25^27]. The mechanisms of osmoprotection in Salmonella remains to be clari¢ed by the genetic analysis of the strains described in this study.
Acknowledgements This work was supported by a Grant-in Aid for Scienti¢c Research (00001987-01) from the Japan Society for the Promotion of Science (JSPS), and by a grant for the Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare.
References [1] Saito, A., Otsuka, K., Hamada, Y., Ono, K. and Masaki, H. (2000) E¡ects of Temperature, pH and sodium chloride concentration on the growth of Salmonella Oranienburg and Salmonella Chester isolated from dried squid and behaviors of those strains in the dried squid. Jpn. J. Food Microbiol. 17, 11^17. [2] Aguilera, J.M., Karel, M. (1997) Preservation of biological materials under desiccation. Crit. Rev. Food Sci. Nutr. 37, 287^309. Review. [3] Glass, K.A., Loe¡elholz, J.M., Ford, J.P. and Doyle, M.P. (1992) Fate of Escherichia coli O157:H7 as a¡ected by pH or sodium chloride and in fermented, dry sausage. Appl. Environ. Microbiol. 58, 2513^2516. [4] Guraya, R., Frank, J.F. and Hassan, A.N. (1998) E¡ectiveness of salt, pH, and diacetyl as inhibitors for Escherichia coli O157:H7 in dairy foods stored at refrigeration temperatures. J. Food Prot. 61, 1098^1102. [5] Masuda, S., Hara-Kudo, Y. and Kumagai, S. (1998) Reduction of Escherichia coli O157:H7 populations in soy sauce, a fermented seasoning. J. Food Prot. 61, 657^661. [6] Tsuji, H. and Hamada, K. (1999) Outbreak of salmonellosis caused by ingestion of cuttle¢sh chips contaminated by both Salmonella Chester and Salmonella Oranienburg. Jpn. J. Infect. Dis. 52, 138^139. [7] Butzler, J.P. and Oosterom, J. (1991) Campylobacter: pathogenicity and signi¢cance in foods. Int. J. Food. Microbiol. 12, 1^8. [8] D’Aoust, J.Y., Sewell, A.M. and Greco, P. (1994) Detection of Salmonella in dry foods using refrigerated pre-enrichment and enrichment broth cultures: summary of collaborative study. J. AOAC Int. 77, 1490^1491. [9] Kim, Y.R., Czajka, J. and Batt, C.A. (2000) Development of a £uorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk. Appl. Environ. Microbiol. 66, 1453^1459. [10] Kunene, N.F., Hastings, J.W. and von Holy, A. (1999) Bacterial populations associated with a sorghum-based fermented weaning cereal. Int. J. Food Microbiol. 49, 75^83. [11] Norton, D.M., McCamey, M.A., Gall, K.L., Scarlett, J.M., Boor, K.J. and Wiedmann, M. (2001) Molecular studies on the ecology of Listeria monocytogenes in the smoked ¢sh processing industry. Appl. Environ. Microbiol. 67, 198^205. [12] Tilden Jr., J., Young, W., McNamara, A.M., Custer, C., Boesel, B., Lambert-Fair, M.A., Majkowski, J., Vugia, D., Werner, S.B., Hol-
FEMSLE 10508 12-6-02
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
[13]
[14]
[15]
[16]
[17]
[18]
lingsworth, J. and Morris Jr., J.G. (1996) A new route of transmission for Escherichia coli : infection from dry fermented salami. Am. J. Public Health 86, 1142^1145. Barcina, I., Lebaron, P. and Vives-Rego, J. (1997) Survival of allochthonous bacteria in aquatic systems: a biological approach. FEMS Microbiol. Ecol. 23, 1^9. Choa, J.C. and Kim, S.J. (1999) Viable, but non-culturable, state of a green £uorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water. FEMS Microbiol. Lett. 170, 257^264. Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: Implications for release of genetically engineered microorganisms. Bio/Technology 3, 817^ 820. Makino, S.-I., Kii, T., Asakura, H., Shirahata, T., Ikeda, T., Takeshi, K. and Itoh, K. (2000) Does enterohaemorrhagic Escherichia coli O157 enter the VNC state in salmon roe? Appl. Environ. Microbiol. 66, 5536^5539. Mattick, K.L., Jorgensen, F., Legan, J.D., Cole, M.B., Porter, J., Lappin-Scott, H.M. and Humphrey, T.J. (2000) Survival and ¢lamentation of Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 at low water activity. Appl. Environ. Microbiol. 66, 1274^1279. Rollins, D.M. and Colwell, R.R. (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl. Environ. Microbiol. 52, 531^538.
93
[19] Santo Domingo, J.W., Harmon, S. and Bennett, J. (2000) Survival of Salmonella species in river water. Curr. Microbiol. 40, 409^417. [20] Gautom, R.K. (1997) Rapid pulsed-¢eld gel electrophoresis protocol for typing of Escherichia coli O157: H7 and other Gram-negative organisms in 1 day. J. Clin. Microbiol. 35, 2977^2980. [21] Nebe-von-Caron, G., Stephens, P.J., Hewitt, C.J., Powell, J.R. and Badley, R.A. (2000) Analysis of bacterial function by multi-colour £uorescence £ow cytometry and single cell sorting. J. Microbiol. Methods 42, 97^114. [22] Kim, W.S., Ren, J. and Dunn, N.W. (1999) Di¡erentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses. FEMS Microbiol. Lett. 171, 57^ 65. [23] Blivet, D., Salvat, G., Humbert, F. and Colin, P. (1997) Evaluation of a new enrichment broth for the isolation of Salmonella spp. from poultry products. Int. J. Food Microbiol. 38, 211^216. [24] Reissbrodt, R., Heier, H., Tscha«pe, H., Kingsley, R.A. and Williams, P.H. (2000) Resuscitation by ferrioxamine E of stressed Salmonella enterica serovar Typhimurium from soil and water microcosms. Appl. Environ. Microbiol. 66, 4128^4130. [25] Cheville, A.M., Arnold, K.W., Buchrieser, C., Cheng, C.M. and Kaspar, C.W. (1996) rpoS regulation of acid, heat, and salt tolerance in Escherichia coli O157:H7. Appl. Environ. Microbiol. 62, 1822^1824. [26] Csonka, L.N. (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol. Rev. 53, 121^147. [27] Wood, J.M. (1999) Osmosensing by bacteria : signals and membranebased sensors. Microbiol. Mol. Biol. Rev. 63, 230^262.
FEMSLE 10508 12-6-02
www.fems-microbiology.org
Passage in mice causes a change in the ability of Salmonella enterica serovar Oranienburg to survive NaCl osmotic stress: resuscitation from the viable but non-culturable state Hiroshi Asakura a , Sou-ichi Makino a; , Tsuyoshi Takagi a , Asumi Kuri a , Takayuki Kurazono b , Masahisa Watarai a , Toshikazu Shirahata a a
Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555 Japan b Saitama Prefectural Institute of Public Health, Saitama 338-0824 Japan Received 6 February 2002; received in revised form 24 April 2002 ; accepted 26 April 2002 First published online 29 May 2002
Abstract An outbreak caused by dried processed squids contaminated with Salmonella Oranienburg occurred in Japan in 1999. Isolates obtained from the causative food were resistant to NaCl osmotic stress, but isolates from the patients were sensitive to NaCl. Although strains from both sources were almost identical in their virulence in mice, a NaCl-resistant strain from food (Sa9911T) became NaCl-sensitive after passage in mice and a NaCl-sensitive strain from one patient (Sa99004) retained NaCl sensitivity after such passage. When dried squid was contaminated experimentally with both strains during processing, only Sa9911T was recovered directly from the final product. Nevertheless, the viability of the Sa99004 cells was over 90% found by fluorescent staining. We suggested that Sa99004 might become viable but non-culturable (VNC) by NaCl stress. This hypothesis was confirmed by resuscitation by efficient enrichment. We concluded that VNC S. Oranienburg would be potentially dangerous contaminants of NaCl-preserved foods and that measures to ensure their detection should be taken at the time of food inspection. 8 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Salmonella Oranienburg; Dried squid; NaCl-resistance ; Viable but non-culturable
1. Introduction The preservation of foods by concentrated NaCl followed by desiccation has been used widely as an e¡ective means of preventing food borne infections [1^5]. However, a di¡use outbreak caused by dried squid, which was contaminated with Salmonella enterica subsp. enterica serovar Oranienburg, occurred in 46 prefectures in Japan in 1999. In the outbreak, over 1500 people su¡ered septicemia and gastroenteritis [1,6]. As all the causative foods were manufactured by the same company, the raw squid was probably contaminated with S. Oranienburg during the production process. This led us to hypothesize that S. Oranienburg can survive and retain its pathogenicity for humans in concentrated NaCl and during desiccation.
* Corresponding author. Tel. : +81 (155) 49 5386; Fax : +81 (155) 49 5386. E-mail address : [email protected] (S.-i. Makino).
Several bacterial pathogens, such as Campylobacter jejuni, Listeria monocytogenes, Escherichia coli and Bacillus cereus, have been detected in dried foods [2,7^12]. In addition, Vibrio cholerae, E. coli O157:H7, C. jejuni and Salmonella spp. become viable but non-culturable (VNC) in environments such as seawater, ground water, rivers and salted food [13^19]. This may result in an underestimation of the true number of viable cells in foodstu¡. In this study, we attempted to clarify how and why S. Oranienburg cells survive under harsh conditions. We showed that the isolates from food remained viable after soaking in NaCl and during dehydration, whereas the isolates from patients did not. Sensitivity to NaCl stress was con¢rmed as the phenotype responsible for the di¡erence in survival rates between these isolates, rather than low water activity or temperature. The importance of the bacterial VNC state during manufacturing processes, the resuscitation of VNC cells, and the potential risk they pose to humans are discussed.
0378-1097 / 02 / $22.00 8 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 1 1 - 5
FEMSLE 10508 12-6-02
88
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
2.4. Survival of S. Oranienburg strains in a NaCl solution
2. Materials and methods 2.1. Salmonella strains and media Nine strains of S. Oranienburg associated with the outbreak and two strains originating from sporadic cases were used in this study (Table 1). The estimated water activity of the dried squid was 0.6 [1]. The liquid culture media included nutrient broth (NB; Difco) lacking NaCl, Hajna tetrathionate broth (HTB ; Eiken, Japan), and brain heart infusion broth (BHI; Difco). Agar plates were made from desoxycholate hydrogen sul¢de lactose agar (DHL; Eiken) and trypticase soy agar (TSA ; BBL/Becton Dickinson). 2.2. Analysis by pulsed ¢eld gel electrophoresis (PFGE) Fingerprinting of bacterial DNA by PFGE was done mainly by the method of Gautom et al. [20] with the following modi¢cations. Each chromosomal DNA sample was digested with 30 U of XbaI or BlnI (TOYOBO) and the resultant fragments were analyzed by electrophoresis through a 1% agarose gel (Amersham) in 0.5UTris^borate^EDTA bu¡er, using Gene Navigator (Amersham). 2.3. Sensitivity to drying Bacterial cells grown at 37‡C for 20 h in NB were washed once with sterile saline, then were resuspended in an identical volume of sterile saline. A 50-Wl aliquot of the suspension was mixed with 450 Wl of sterile saline in each well of a 48-well microtiter plate (Nalge Nunc). The plate was incubated uncovered at 45‡C for 24 h to dry the suspension. Sterile water (500 Wl) was then added to each well, and 0.1 ml of the resultant suspension was spread on DHL and TSA plates after serial dilution to measure the colony forming units (CFU).
Table 1 S. Oranienburg strains used in this study Strains
Origin
Symptoms
Sa99001 Sa99004 Sa99026 Sa99061 Sa99073 Sa9911T Sa9912A Sa9921K Sa9937O Sai-1 Sai-2
Patient patient patient patient patient dried squid raw materials raw materials raw materials patient patient
asymptomatic septicemia septicemia septicemia asymptomatic ^ ^ ^ ^ septicemia diarrhea
Approximately 108 ^109 S. Oranienburg cells grown at 37‡C for 20 h in NB were suspended in 10 ml of NaCl solution at various concentrations and were incubated at 37‡C for 24 h. A 0.1-ml aliquot of the suspension and its serial dilutions were spread on DHL and TSA plates to measure the CFU. To ensure the data were accurate, the experiment was repeated in triplicate. 2.5. Experimental infection of mice with S. Oranienburg A total of 50 BALB/c female mice aged 6 weeks were injected intraperitoneally with 6.6U105 S. Oranienburg cells grown at 37‡C for 18 h in NB. At 1 day after infection and then weekly for up to 4 weeks, ¢ve mice were killed with chloroform to collect their liver, spleen, blood, and intestinal contents. Tissue samples were weighed and homogenized in 1 ml PBS. Aliquots of the suspensions were spread on DHL plates to measure the CFU. 2.6. Recovery of Salmonella cells from experimentally infected dried squid 50 ml of 7% NaCl solution containing 5 g of raw squid was inoculated with 6.6U109 S. Oranienburg cells were grown at 37‡C for 18 h in NB, and were incubated at 4‡C for 24 h. The squid was removed, dried at 45‡C for 24 h, soaked in 5 ml bu¡ered peptone water (BPW, pH 7.2), and incubated at 37‡C for 1 h. Aliquots (0.1 ml) of serially diluted samples were spread on DHL plates to measure the CFU. At the same time, 1 ml of the suspension and its serial dilutions were inoculated in 10 ml of HTB, and were incubated at 37‡C for 20 h to isolate Salmonella cells and to calculate the most probable number (MPN) of these cells. Another enrichment procedure using ¢lter-sterilized NB culture supernatant from an overnight culture of strain Sa9911T or Sa99004 was used, essentially by the method of Santo Domingo et al. [19]. 1 ml of the BPW suspension containing squid and its serial dilutions were added to 10 ml of 5% BHI containing 100 Wl of the supernatant, followed by incubation for 7 days at room temperature. To examine the e¡ect of the supernatant on enrichment, 5% BHI without the supernatant was used as a control. Aliquots of the cultures were spread on DHL plates to isolate Salmonella cells and to measure the MPN. 2.7. Determination of viability
Nine strains except Sai-1 and Sai-2 were associated with an outbreak caused by dried squid. The other two strains were isolated from sporadic outbreaks.
Salmonella cells in BPW suspension with squid and in NaCl solutions were stained by using the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes). The bacteria were examined under £uorescence microscopy and the viability of 1000 cells was assessed.
FEMSLE 10508 12-6-02
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
89
3.2. Sensitivity of S. Oranienburg strains to drying or NaCl stress
Fig. 1. PFGE patterns of S. Oranienburg strains. Five representative strains containing two S. Oranienburg strains, Sai-1 and Sai-2, isolated from other sporadic outbreaks, were analyzed by PFGE using BlnI (lanes 1^5) and XbaI (lanes 6^10). Lanes 1 and 6, Sai-1; 2 and 7, Sai-2; 3 and 8, Sa99004; 4 and 9, Sa9911T; 5 and 10; Sa9921K.
3. Results 3.1. DNA analysis of S. Oranienburg strains Epidemiological studies [1,6] have suggested that S. Oranienburg isolates from patients in the outbreak were the same as isolates from the causative food, dried squid. To con¢rm the relationship, nine isolates listed in Table 1 were examined genetically. As expected, their PFGE patterns were identical (Fig. 1, lanes 3^5 and 8^10), but differed from two strains from unrelated sporadic cases (Fig. 1, lanes 1, 2, 6 and 7).
Nine isolates from the outbreak were suspended in saline to ¢nd their sensitivity to drying. First, the suspensions were maintained at 45‡C for 24 h, but no marked di¡erence was observed in survival rates between isolates from food and from patients (Table 2). After drying in saline, however, the CFU of ¢ve strains from patients decreased markedly, especially strain Sa99004 formed no colonies after drying, but four strains from food survived in large numbers and Sa9911T was the most resistant to drying (Table 2), resulting in strains from patients being sensitive to drying in NaCl while those from food were resistant. However, when their sensitivity to drying was examined in sterile water instead of saline, all the isolates appeared to be resistant (Table 2). Therefore, we concluded that sensitivity to drying was concurrent with sensitivity to NaCl. The changes in CFU of each strain in various NaCl concentrations was examined. With increasing NaCl concentrations, the number of the remaining four strains from patients declined more slowly than Sa99004. However, the decline was greater than that of the strains from food (Fig. 2). Thus, strains from food were resistant to NaCl stress, but those from patients were sensitive. The NaCl sensitivity-resistance phenotype of these strains remained stable, even though they were subcultured in NB for 100 passages (data not shown). The CFU of strains from food decreased by approximately 100-fold after incubation for 24 h in 17% NaCl, whereas strain Sa99004 of patient origin formed no colonies after 24 h in 7% NaCl (Fig. 2). Almost identical results were obtained by using DHL and TSA plates for the growth of S. Oranienburg cells after they were incubated in NaCl solution (data not shown). 3.3. Pathogenicity of S. Oranienburg strains in mice As the NaCl sensitivity of S. Oranienburg isolates from
Table 2 Survival of S. Oranienburg isolates through the drying process Strains
Patient origin Sa99001 Sa99004 Sa99026 Sa99061 Sa99073 Food origin Sa9911T Sa9912A Sa9921K Sa9937O
Culturable cell numbers (CFU ml31 ) Initial dose
Drying in saline
Drying in water
Incubation in salinea
1.7U109 5.3U108 4.4U108 6.8U108 3.8U108
3.5U102 6 10 1.9U102 2.4U102 1.5U102
6.9U106 1.8U107 2.2U107 1.4U106 2.5U107
2.6U107 2.2U107 1.7U107 2.8U107 1.2U107
5.6U108 3.7U108 5.0U108 9.4U108
2.8U107 8.9U105 2.3U106 7.2U106
3.0U106 1.0U107 5.6U106 1.4U107
1.8U107 2.5U107 2.2U107 2.7U107
a
All isolates were incubated in saline at 45‡C for 24 h. Each value shows the standard values in the experiments done in triplicate.
FEMSLE 10508 12-6-02
90
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
Fig. 2. Bacterial cell numbers of S. Oranienburg in NaCl solution. The values are the standard means of three experiments.
food and patients were di¡erent, the fact that their pathogenicity might also di¡er was plausible. This possibility was examined in mice by intraperitoneal injection of 6.6U105 CFU of strains Sa9911T and Sa99004, which were selected as the representative isolates from food and patient, respectively. For at least 40 days, all mice survived with no symptoms. In the CFU of bacteria recovered from liver, spleen, blood and intestinal contents, there was the two groups of mice showed no signi¢cant di¡erence for up to 4 weeks after injection (Fig. 3). In this experiment, no signi¢cant di¡erence was detected in pathogenicity for mice. The pathogenicity of NaCl-stressed isolates was compared; NaCl-stressed Sa9911T cells were recovered from organs at similar numbers (Fig. 3), but NaCl-stressed Sa99004 cells were not recovered. This was consistent with the observation that NaCl-stressed Sa99004 cells did not form any colonies on the plates (Fig. 2). 3.4. Phenotypic change in NaCl sensitivity by passage in animals A total of 50 colonies were selected from mice infected with S. Oranienburg strains, and their sensitivity to NaCl
was examined by testing their resistance to drying. Of the Salmonella colonies recovered from mice infected with the NaCl-resistant strain, 4^88% Sa9911T were sensitive to NaCl during passage in vivo, but all the colonies derived from the NaCl-sensitive strain Sa99004 remained phenotypically unchanged. The NaCl resistance-sensitivity phenotype of the colonies derived from Sa9911T recovered from mice remained stable, even though they were subcultured in NB for 100 passages (data not shown). 3.5. NaCl sensitivity of S. Oranienburg in experimentally processed dried squid When the strains from patients were incubated in 17% NaCl solution, the suspensions contained viable cells (Fig. 2), but they remained sensitive to NaCl (data not shown). However, ¢nding if they become resistant to NaCl after incubation in dried squid was necessary. Strains Sa99004 and Sa9911T (approximately 6.6U109 cells) were incubated separately in 7% NaCl solution containing raw squid at 4‡C for 24 h. The squid was then processed experimentally. Each dried squid was soaked in BPW at 37‡C for 1 h, and then 0.1 ml of the suspension and serial dilutions were spread on DHL plates. Although approximately 4.0U108
FEMSLE 10508 12-6-02
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
91
Fig. 3. Comparison of pathogenicity of S. Oranienburg strains in mice. Strain Sa9911T of food origin (A) and strain Sa99004 of patient origin (B) were intraperitoneally injected into BALB/c mice and the viable bacteria were counted in liver, spleen, peritoneal blood, and feces every week for 4 weeks after infection.
cells were recovered from 1 g of dried squid contaminated with strain Sa9911T; no Sa99004 cells were recovered after the same treatment (Table 3). The BPW suspensions were examined for cell viability by staining with £uorescent dye; over 90% of bacterial cells from food and patients were viable among a total of 1000 bacterial cells selected randomly (data not shown).
richment cultures contained Salmonella cells and no cells were detected by direct plating (Table 3), Sa99004 cells must have entered the VNC state and been resuscitated successfully in both enrichment cultures (Table 3). The sensitivity to NaCl of 50 bacterial colonies, which were recovered from dried squid by enrichment plating, remained stable, even though they were subcultured in NB for 100 passages (data not shown).
3.6. Recovery of VNC Salmonella cells Salmonella cells were recovered by enrichment culture in HTB and the MPN was measured simultaneously. The squid BPW suspension from strain Sa99004 was estimated to contain 0^45 (SD = 17.76) viable cells per ml (Table 3). A second enrichment procedure was done by using the culture supernatant [19]. By this method, the suspension was estimated to contain 225^550 (SD = 187.64) viable cells per ml (Table 3). The culture supernatant of strains Sa9911T and Sa99004 caused resuscitation. As all the en-
4. Discussion A di¡use outbreak caused by dried squid contaminated with S. Oranienburg occurred in Japan in 1999 [1,6]. As the isolates from food and patients showed identical PFGE patterns in this study, we concluded that S. Oranienburg contamination of the food caused the outbreak. Bacterial growth is generally poor in foods with a low water activity of around 0.6 [5], therefore the raw materi-
Table 3 Resuscitation of S. Oranienburg cell suspension from the VNC state Enrichment
Recovery of Sa99004 cells froma
Procedures Direct plating
1 ml
100Wl
10Wl
1Wl
SD
(0/25) + (5/25) + (23/25)
(0/25) + (1/25) + (13/25)
(0/25) 3 (0/25) + (5/25)
(0/25) 3 (0/25) 3 (0/25)
0 0^45 17.76 225^550 187.64
HTB Supernatant
MPN values
a
1 ml and its serial dilutions of the BPW suspensions contaminated with Sa99004 cells with dry-processed squids were used for each experiment as described in Section 2. In a total of 25 tubes, the numbers of Salmonella cells recovered in vitro are shown in parentheses. + and 3 indicate that Salmonella cells can or can not be recovered from the samples, respectively.
FEMSLE 10508 12-6-02
92
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
als of the dried squid appeared to have been highly contaminated with S. Oranienburg before or during processing. Estimation by the MPN method indicated that approximately 10^103 cells of S. Oranienburg were present in each gram of the contaminated food, but only fewer than 20 cells were isolated by direct plating [1]. This level of S. Oranienburg contamination was unlikely to establish septicemia, the main symptom of the patients in this outbreak (Table 1). However, if a large proportion of the bacterial cells in the dried squid had entered the VNC state, a much higher number of viable cells, which are intractable under normal culture methods, could have contaminated the food without being detected and caused the outbreak. Salmonella cells rapidly enter the VNC state in aqueous conditions [14,17,19] and their numbers detected in the dried squid gradually decrease during storage [1]. Thus, precise enumeration of bacteria in food is not easy and predicting if a particular level of contamination results in human infection is extremely di⁄cult. In this study, the sensitivity of the strains from food or patients to drying in saline and in distilled water was tested. Even if the initial NaCl concentration is low, the concentration gradually increases during the drying process. Therefore, the sensitivity of bacteria to drying in saline would re£ect their sensitivity to NaCl. As the resistance and sensitivity to drying was consistent with resistance and sensitivity to NaCl, the tests of sensitivity to drying used in this study would be a simple and useful screening method to predict the NaCl sensitivity of the bacteria. This study showed that strains from food were resistant to NaCl and those from patients were sensitive. The NaClresistant strain from food became sensitive following passage in mice, although the basic mechanism for this observation was not clear. This one-way phenotypic conversion occurred only in vivo, and the NaCl resistance in strains of food origin remained unchanged in vitro. We reported previously an outbreak of food poisoning caused by enterohemorrhagic E. coli (EHEC) O157 in contaminated salted salmon roe [16]. What was observed in this outbreak is consistent with the results of this study. In the outbreak due to contaminated salmon, EHEC O157 isolated from patients were sensitive to NaCl while strains from food were resistant. Furthermore, the NaCl-resistant O157 strain also became NaCl-sensitive after passage in vivo. We suggest that the natural phenotype of enteric pathogens like Salmonella and O157 in the environment is NaCl-resistant. The results of this study showed that the pathogenicity for mice of NaCl-sensitive and -resistant isolates was almost the same, and we conclude that the NaCl sensitivity is not related to the virulence. Under various stressful conditions [13^19,21,22], bacterial cells may become VNC as a protective mechanism. Resuscitation could occur under certain conditions [19,23,24], and hence bacterial cells becoming VNC might
protect against environmental stress. In this study, the resuscitation of Salmonella cells from the VNC state was shown in vitro by two enrichment procedures. These methods might be useful not only to detect Salmonella cells, but also to estimate viable numbers of other bacterial pathogens that have become VNC in foods. The genetic response induced by NaCl osmotic stress has been reported for several bacterial species [25^27]. The mechanisms of osmoprotection in Salmonella remains to be clari¢ed by the genetic analysis of the strains described in this study.
Acknowledgements This work was supported by a Grant-in Aid for Scienti¢c Research (00001987-01) from the Japan Society for the Promotion of Science (JSPS), and by a grant for the Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare.
References [1] Saito, A., Otsuka, K., Hamada, Y., Ono, K. and Masaki, H. (2000) E¡ects of Temperature, pH and sodium chloride concentration on the growth of Salmonella Oranienburg and Salmonella Chester isolated from dried squid and behaviors of those strains in the dried squid. Jpn. J. Food Microbiol. 17, 11^17. [2] Aguilera, J.M., Karel, M. (1997) Preservation of biological materials under desiccation. Crit. Rev. Food Sci. Nutr. 37, 287^309. Review. [3] Glass, K.A., Loe¡elholz, J.M., Ford, J.P. and Doyle, M.P. (1992) Fate of Escherichia coli O157:H7 as a¡ected by pH or sodium chloride and in fermented, dry sausage. Appl. Environ. Microbiol. 58, 2513^2516. [4] Guraya, R., Frank, J.F. and Hassan, A.N. (1998) E¡ectiveness of salt, pH, and diacetyl as inhibitors for Escherichia coli O157:H7 in dairy foods stored at refrigeration temperatures. J. Food Prot. 61, 1098^1102. [5] Masuda, S., Hara-Kudo, Y. and Kumagai, S. (1998) Reduction of Escherichia coli O157:H7 populations in soy sauce, a fermented seasoning. J. Food Prot. 61, 657^661. [6] Tsuji, H. and Hamada, K. (1999) Outbreak of salmonellosis caused by ingestion of cuttle¢sh chips contaminated by both Salmonella Chester and Salmonella Oranienburg. Jpn. J. Infect. Dis. 52, 138^139. [7] Butzler, J.P. and Oosterom, J. (1991) Campylobacter: pathogenicity and signi¢cance in foods. Int. J. Food. Microbiol. 12, 1^8. [8] D’Aoust, J.Y., Sewell, A.M. and Greco, P. (1994) Detection of Salmonella in dry foods using refrigerated pre-enrichment and enrichment broth cultures: summary of collaborative study. J. AOAC Int. 77, 1490^1491. [9] Kim, Y.R., Czajka, J. and Batt, C.A. (2000) Development of a £uorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk. Appl. Environ. Microbiol. 66, 1453^1459. [10] Kunene, N.F., Hastings, J.W. and von Holy, A. (1999) Bacterial populations associated with a sorghum-based fermented weaning cereal. Int. J. Food Microbiol. 49, 75^83. [11] Norton, D.M., McCamey, M.A., Gall, K.L., Scarlett, J.M., Boor, K.J. and Wiedmann, M. (2001) Molecular studies on the ecology of Listeria monocytogenes in the smoked ¢sh processing industry. Appl. Environ. Microbiol. 67, 198^205. [12] Tilden Jr., J., Young, W., McNamara, A.M., Custer, C., Boesel, B., Lambert-Fair, M.A., Majkowski, J., Vugia, D., Werner, S.B., Hol-
FEMSLE 10508 12-6-02
H. Asakura et al. / FEMS Microbiology Letters 212 (2002) 87^93
[13]
[14]
[15]
[16]
[17]
[18]
lingsworth, J. and Morris Jr., J.G. (1996) A new route of transmission for Escherichia coli : infection from dry fermented salami. Am. J. Public Health 86, 1142^1145. Barcina, I., Lebaron, P. and Vives-Rego, J. (1997) Survival of allochthonous bacteria in aquatic systems: a biological approach. FEMS Microbiol. Ecol. 23, 1^9. Choa, J.C. and Kim, S.J. (1999) Viable, but non-culturable, state of a green £uorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water. FEMS Microbiol. Lett. 170, 257^264. Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: Implications for release of genetically engineered microorganisms. Bio/Technology 3, 817^ 820. Makino, S.-I., Kii, T., Asakura, H., Shirahata, T., Ikeda, T., Takeshi, K. and Itoh, K. (2000) Does enterohaemorrhagic Escherichia coli O157 enter the VNC state in salmon roe? Appl. Environ. Microbiol. 66, 5536^5539. Mattick, K.L., Jorgensen, F., Legan, J.D., Cole, M.B., Porter, J., Lappin-Scott, H.M. and Humphrey, T.J. (2000) Survival and ¢lamentation of Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 at low water activity. Appl. Environ. Microbiol. 66, 1274^1279. Rollins, D.M. and Colwell, R.R. (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl. Environ. Microbiol. 52, 531^538.
93
[19] Santo Domingo, J.W., Harmon, S. and Bennett, J. (2000) Survival of Salmonella species in river water. Curr. Microbiol. 40, 409^417. [20] Gautom, R.K. (1997) Rapid pulsed-¢eld gel electrophoresis protocol for typing of Escherichia coli O157: H7 and other Gram-negative organisms in 1 day. J. Clin. Microbiol. 35, 2977^2980. [21] Nebe-von-Caron, G., Stephens, P.J., Hewitt, C.J., Powell, J.R. and Badley, R.A. (2000) Analysis of bacterial function by multi-colour £uorescence £ow cytometry and single cell sorting. J. Microbiol. Methods 42, 97^114. [22] Kim, W.S., Ren, J. and Dunn, N.W. (1999) Di¡erentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses. FEMS Microbiol. Lett. 171, 57^ 65. [23] Blivet, D., Salvat, G., Humbert, F. and Colin, P. (1997) Evaluation of a new enrichment broth for the isolation of Salmonella spp. from poultry products. Int. J. Food Microbiol. 38, 211^216. [24] Reissbrodt, R., Heier, H., Tscha«pe, H., Kingsley, R.A. and Williams, P.H. (2000) Resuscitation by ferrioxamine E of stressed Salmonella enterica serovar Typhimurium from soil and water microcosms. Appl. Environ. Microbiol. 66, 4128^4130. [25] Cheville, A.M., Arnold, K.W., Buchrieser, C., Cheng, C.M. and Kaspar, C.W. (1996) rpoS regulation of acid, heat, and salt tolerance in Escherichia coli O157:H7. Appl. Environ. Microbiol. 62, 1822^1824. [26] Csonka, L.N. (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol. Rev. 53, 121^147. [27] Wood, J.M. (1999) Osmosensing by bacteria : signals and membranebased sensors. Microbiol. Mol. Biol. Rev. 63, 230^262.
FEMSLE 10508 12-6-02