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Applied and Environmental Microbiology, December 1998, p. 4658-4662, Vol. 64, No. 12
Department of Microbiology, Montana State
University, Bozeman, Montana 59717,1 and
Thames Water Utility, Reading RG2 0JN, United
Kingdom2
Received 23 April 1998/Accepted 14 September 1998
Escherichia coli O157:H7 can persist for days to weeks
in microcosms simulating natural conditions. In this study, we used a
suite of fluorescent, in situ stains and probes to assess the influence
of starvation on physiological activity based on membrane potential
(rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity
(5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular
esterase activity (ScanRDI assay), and 16S rRNA content.
Growth-dependent assays were also used to assess substrate
responsiveness (direct viable count [DVC] assay), ATP activity
(MicroStar assay), and culturability (R2A agar assay). In addition,
resistance to chlorine disinfection was assessed. After 14 days of
starvation, the DVC values decreased, while the values in all other
assays remained relatively constant and equivalent to each other.
Chlorine resistance progressively increased through the starvation
period. After 29 days of starvation, there was no significant
difference in chlorine resistance between control cultures that had not
been exposed to the disinfectant and cultures that had been exposed.
This study demonstrates that E. coli O157:H7 adapts to
starvation conditions by developing a chlorine resistance phenotype.
Escherichia coli is a
facultatively anaerobic commensal member of the intestinal microflora
of humans and other warm-blooded animals. Strains associated with
hemorrhagic colitis and bloody diarrhea are placed in the
enterohemorrhagic E. coli group, which includes the O157:H7
serotype. From its initial isolation and description in 1982 (40), E. coli O157:H7 has been implicated in
numerous outbreaks in the United States and Great Britain. The most
notable of these outbreaks have been associated with the consumption of
contaminated beef products (10, 51). However, other vehicles
of transmission have been described and include person-to-person
contact and consumption of vegetables (3), untreated water
(drinking water and lake water) (48), and unpasteurized milk
and apple cider (10), as well as swimming in freshwater lakes (1). Nonhuman reservoirs have been identified and
include beef and dairy cattle (9, 16), sheep
(21), and wild birds (50).
Previous studies have shown that E. coli O157:H7 remains
culturable for days to weeks in cattle feces, soil, and different types
of water (28, 36). In this study we used a suite of fluorogenic stains and probes to assess physiological activity and
compared the data obtained to culturability during a 14-day starvation
period. In addition, the ability of starved cells to resist
disinfection by hypochlorous acid (chlorine) was determined.
Bacterial strains and culture conditions.
E. coli
O157:H7 strain 932 was provided by the U.S. Environmental Protection
Agency. Cultures of E. coli O157:H7 frozen at Total viable plate counts.
Samples were diluted in sterile
reagent grade water (Milli-Q UV Plus; Millipore Corp., Bedford, Mass.)
and used to inoculate agar plates by a modified drop plate method
(19). Each day for 3 days total viable plate counts were
obtained from R2A agar plates after incubation at 20 to 23°C
(39). R2A agar plate counting associated with 16S rRNA data
was not performed in parallel with the other assays described below.
Therefore, the R2A agar plate count data cannot be directly compared to
the other data obtained.
Total cell count and volume determinations.
Total cell
counts were determined after 1.0 ml of a sample was fixed with 30 µl
of formalin and 250 µl of a 4',6-diamidino-2-phenylindole (DAPI)
solution (25 µg/ml; Sigma Chemical Co., St. Louis, Mo.). Samples were
incubated at room temperature for 4 h before they were filtered
onto black polycarbonate membrane filters. Cell volumes were calculated
by using the mean values for length and width obtained from 10 randomly
selected bacteria per filter (37).
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Starvation on Physiological Activity and
Chlorine Disinfection Resistance in Escherichia coli
O157:H7
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C in
dimethyl sulfoxide (4) were used to inoculate 100 ml of YT
broth containing (per liter) 10 g of tryptone, 5 g of yeast
extract, 5 g of glucose, and 5 g of sodium chloride (pH 7.2).
Late-log-phase cultures (after incubation for 16 to 18 h at
25°C) were diluted (approximately 108 cells/ml) in M9
medium to which no carbon source had been added (4).
Starvation cultures in 1-liter flasks containing 200 ml of M9 medium
were inoculated with 1.0 ml of the cell suspension and incubated at 20 to 23°C on a rotary shaker (100 rpm). Four 1.0-ml samples were
removed at the time of inoculation and on days 2, 5, 10, and 14 for
analysis. The disinfection resistance assays were also performed on day 29.
Substrate responsiveness assay.
A modified direct viable
count (DVC) assay was performed as described by Singh et al.
(42) by using nalidixic acid at a final concentration of 10 µg/ml. Cells were considered viable if they were 
1.5 times the
average length of control cells (n = 10) that had not
been exposed to nalidixic acid or chlorine.
Microcolony assay. The presence of ATP was determined by using the MicroStar system (Millipore Corp.), which detects ATP-driven bioluminescence in microcolonies. Each sample was diluted and filtered onto hydrophobic grid membranes (diameter, 47 mm; pore size, 0.45 µm; type RMHV04720; Millipore Corp.). The inoculated membranes were transferred to mHPC (Millipore Corp.) agar plates and incubated for 4 h at 35°C for samples collected at the time of inoculation and for 18 h at 20 to 23°C for samples collected on days 2 to 14. All of the filters were processed for release and detection of ATP as described by the manufacturer.
In situ assays. Aliquots (1.0 ml) of each of the samples collected were vacuum filtered through black polycarbonate membranes (pore size, 0.2 µm; diameter, 25 mm; type GTBP; Millipore Corp.) for each assay. All stains and probes were made as described below, filter sterilized, and applied as 600-µl overlays, and the preparations were incubated at room temperature shielded from light. Control experiments were performed with actively growing cells and cells that had been heat inactivated at 60°C for 1 h. Cells were considered positive in the assays if their fluorescence was greater than that of inactivated control cells.
(i) Assessment of membrane potential. Rhodamine 123 (Rh123) is a lipophilic cationic stain that is preferentially accumulated in cells that have a membrane potential (18). Rh123 (13.0 µM; Sigma) was dissolved in TE (10 mM Tris, 1 mM EDTA; pH 8.0) and incubated for 15 min at 20 to 23°C.
(ii) Assessment of membrane integrity. The live stain (Syto9) from a LIVE/DEAD BacLight viability kit (catalog no. L-7007; Molecular Probes, Eugene, Oreg.) was diluted in reagent grade water as recommended by the manufacturer and incubated for 30 min at 20 to 23°C.
(iii) Assessment of respiratory activity. Intracellular reduction of 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC) (5 mM; PolyScience, Warrington, Pa.) to an insoluble fluorescent formazan by dehydrogenases was used to assess respiratory activity (43). The CTC was dissolved in reagent grade water and incubated for 4 h at 20 to 23°C.
(iv) Assessment of esterase activity. Esterase activity was determined by using a ScanRDI solid-phase cytometer (32). Diluted samples were filtered onto black polycarbonate filters and subsequently placed on saturated pads containing the fluorogenic esterase substrate ChemChrome V3 (diluted 1/100 in ChemSol B1). The filters were incubated for 30 min at 30°C.
(v) 16S rRNA content. A polyamide nucleic acid probe (12) was designed and labeled at its 5' end with biotin and an N-terminal lysine for detection of E. coli (University of Southampton, Southampton, United Kingdom). The probe was dissolved in 10% trifluoroacetic acid and heated to 50°C before use. Samples were filtered through metallized membrane filters (pore size, 0.45 µm; Corning Costar Corp., Cambridge, Mass.), fixed with 6% paraformaldehyde (20 min at 23°C), and rinsed with sterile water, and the retained cells were lysed with lysozyme (50 µg/ml; 10 min at 23°C). The lysed cells were rinsed with water and subsequently hybridized for 30 min at 50°C in buffer (2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4; pH 7.2) containing 10 µmol of the probe. The membranes were washed with a second buffer (20 mM Tris-HCl, 180 mM NaCl, 5 mM EDTA, 0.01% sodium dodecyl sulfate; pH 7.2) for 15 min at 50°C, and this was followed by three washes with water at 23°C. The hybridized cells were detected by using a tyramide signal amplification kit (TSA-Direct GreenFISH; NEN Life Science Products, Boston, Mass.) (41). The number of fluorescent cells per filter was calculated from digital images (20 images per filter), and the data were analyzed by using image analysis software (Digital Pixel, Brighton, United Kingdom).
Disinfection assay. One milliliter of a starvation culture was gently mixed with 100 ml of reagent grade water (pH 6.8, 20 to 23°C). To prepare the nonchlorinated control sample, 4 ml was transferred to a tube containing sodium thiosulfate (0.8 mM). A standardized sodium hypochlorite solution (catalog no. 55290; Fisher Scientific Co., St. Louis, Mo.) was added to the remaining cell suspension to a final concentration of 0.5 ppm (as free chlorine), and the preparation was shaken at 100 rpm for 5 min at 20 to 23°C. Following this treatment, 4 ml was transferred to a tube containing sodium thiosulfate. Both samples were diluted in reagent grade water and plated onto R2A agar, tryptone-lactose-yeast extract agar (TLY), and TLY supplemented with 0.1% deoxycholate (TLYD). All plates were incubated in the dark at 20 to 23°C and counted each day for 3 days.
The percentage of disinfectant-resistant (%R) cells was expressed as follows: %R = (100 %I), where %I is the ratio of
sublethally injured colony counts to noninjured colony counts {i.e.,
[(TLY counts TLYD counts)/TLY counts] × 100} (42,
52). When the TLYD counts exceeded the TLY counts, injury was
assigned a value of zero.
Statistical evaluation. Cell and plate counts were log10 transformed, and the means and standard errors of the means were calculated. The calculated means for each assay for each time point were compared by using a one-way analysis of variance (P = 0.05) (14). Data sets with a significant analysis of variance value were analyzed further to determine which means within the data set were significantly different; the Tukey-Kramer method was used to calculate minimum significant differences (14). All data evaluations were performed by using Minitab, release 11.12 (Minitab, Inc., State College, Pa.).
Nucleotide sequence accession number. The probe sequence used in this study was obtained from the EMBL Data Library (accession no. X80724). This sequence corresponds to the sequence at E. coli positions 71 to 86 (5' GCA AAG CAG CAA GCT C 3').
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Following 2 days of incubation, cell biovolumes reached minimum values and remained relatively constant for the following 12 days, indicating that a transition from the stationary phase to a starvation state had occurred (Table 1). Concurrently, the cell numbers increased and then remained constant during the same time interval (Table 1).
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Effects of starvation on growth-dependent assays. Three of the assays used in this study (the R2A agar, DVC, and MicroStar assays) are dependent on a cell's ability to respond to the presence of nutrients, and subsequent growth is required for detection and assessment of physiological activity. After 2 days of starvation, the R2A agar plate, DVC cell and MicroStar microcolony counts increased and then remained relatively constant through day 10 (Table 2). By day 14 the DVC cell counts had decreased to a value that was insignificantly lower than the values determined by the other two assays.
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Effects of starvation on membrane integrity and potential. Membrane integrity and potential followed similar trends, as demonstrated by previously described assays (Table 2). There was no significant difference among the LIVE/DEAD Bac-Light assay, the intracellular accumulation of Rh123, and the reference assays (i.e., total cell and viable plate count assays).
Effects of starvation on selected enzyme activity. The following two types of endogenous enzyme activity were assessed during this study: dehydrogenase activity (CTC assay) and esterase activity (ChemChrome V3 assay) (Table 2). The CTC counts were significantly lower only on day 2 of the experiment compared to the results of other assays. The two assays were equivalent in their counts for the remainder of the starvation period.
Effects of starvation on 16S rRNA content. The relatively stable R2A agar and hybridized cell counts from day 2 through day 14 (Table 3) followed similar trends, as observed with the other assays (Table 2). From the time of inoculation through day 10, the 16S rRNA-based cell counts were on average 2.1 times greater than the R2A agar plate counts. The difference increased to 4.2 times greater by day 14. The 16S rRNA-based cell counts were insignificantly less than or equal to the total cell counts throughout the starvation period (Table 2).
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Effects of starvation on disinfection resistance. The starvation conditions used in this study promoted the development of a phenotype that was resistant to sublethal injury induced by membrane-active detergents (e.g., deoxycholate). This effect was demonstrated by the significant decrease in the percentage of injured cells from the time of inoculation to day 14 (Fig. 1). Concomitantly, there was an increase in resistance to chlorine injury (Table 4). The percentage of injury-resistant cells that had been starved but not exposed to disinfectant increased by approximately 36% from the time of inoculation to day 5 and then remained relatively constant through day 29. When parallel samples were exposed to hypochlorous acid, injury resistance increased by approximately 51% from the time of inoculation to day 5 and then progressively increased another 31% by day 29.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A limited number of studies have evaluated the ability of E. coli O157:H7 to persist in natural environments. Geldreich et al. (15) inoculated filter-sterilized drinking water from a distribution system in which an E. coli O157:H7 outbreak had occurred with a strain of the isolated pathogen. After 11 days of incubation at 5°C, there was no significant decrease in culturability. After 35 days, there was only a 1.4-log decrease in culturability. Maule (28) inoculated several types of microcosms to assess the persistence of E. coli O157:H7 in cattle farm environments. Plate counts were reduced to below the detection limit in cattle slurry (9 days), by approximately 2.5 logs in cattle feces (54 days), by 1 log in soil cores (63 days), and by 5 logs in river water (13 days). Porter et al. (36) found that E. coli O157:H7 culturability was reduced by only 0.8 and 0.5 log in sterile and untreated pond water (20 days), respectively. The differences in culturability among these studies may be partially attributed to differences in medium formulations and selectivity, since different MacConkey-based agars were used for primary isolation. The fact that enteropathogenic bacteria cannot be consistently recovered with media containing selective components following exposure to aquatic stresses has been demonstrated previously (29). In this study we avoided the problems associated with culturability by using intracellular fluorescent stains and probes to assess physiological activity at the single-bacterium level.
Total cell biovolume was used to determine if and when the isolate which we studied entered the starvation phase of its life cycle (Table 1). Between the time of inoculation and day 5 not only was there a decrease in cell biovolume, but there was also an increase in cell number. These responses resulted in a significant increase (approximately 92%) in total biovolume. However, there was no significant change in biovolume from day 5 to day 14. These data suggest that the isolate of E. coli O157:H7 used ultimately compensated for the reduction in nutrients by increasing the cell surface-to-volume ratio through reductive division, a common response in starved bacterial cells (33).
The stains and probes used in this study showed that after an initial
increase in biovolume, the physiological activity and membrane
integrity of E. coli O157:H7 remained relatively constant during the remainder of the 2-week starvation period (i.e., from day 5 to day 14) (Table 2). In addition, there was no detectable decrease in
culturability on R2A agar during the starvation period (Table 2).
However, the 16S rRNA cell counts were consistently greater than the
corresponding R2A agar plate counts (Table 3), suggesting that the
hybridization assay was more sensitive than the culturability assay.
Several studies have attempted to correlate the fluorescence intensity
of hybridized 16S rRNA probes with bacterial viability (20,
38). From these studies it can be generally concluded that rRNA
decay rates and probe intensities differ between and within bacterial
species depending on the growth conditions. It has been estimated that
approximately 81% of the total RNA (approximately 2,100 fg
cell
1) in a bacterial cell at a doubling rate of 2.5 h1 is rRNA (6, 34). The lower detection limit
for RNA in procaryotes when multiple 16S rRNA probes are used
simultaneously has been estimated to be 0.3 fg cell1,
approximately 99% lower than the cellular concentrations during log
phase (24, 25). Therefore, the consistent detection of E. coli O157:H7 cells with 16S rRNA probes during the 14-day
starvation period supports the data obtained in the other in situ
assays, in that the isolate remained physiologically active.
Disinfection resistance has been defined as being inversely proportional to disinfection sensitivity (23). Traditionally, disinfection resistance has been expressed as the difference in culturability on nonselective media before and after exposure to a disinfectant. In the TLY-TLYD plate assay used in this study deoxycholate was included as a selective component of TLYD in order to assess the sensitivity of bacterial membranes to the detergent's disruptive action. We assumed that since exposure to chlorine has been shown to damage cell membranes, to alter associated functions, and to inhibit colony formation (5, 8, 30, 31, 49), it also enhances the ability of deoxycholate to disrupt injured membranes, leading to a lack of culturability on TLYD. We propose that this method for assessing sensitivity (or resistance) to chlorine is appropriate, since the site of sublethal damage by this disinfectant is the bacterial membrane. Therefore, the use of deoxycholate-based media provides an indirect method for assessing chlorine resistance relative to membrane-associated injury.
Starvation alone promoted the development of an injury-resistant membrane structure relative to deoxycholate susceptibility in the absence of disinfection (Fig. 1). Resistance to chlorine injury followed a similar trend, as the difference between the percentage of chlorine-resistant cells before disinfection and the percentage of chlorine-resistant cells after disinfection decreased from 55.2% at the time of inoculation to 8.5% on day 29 (Table 4). Resistance to chlorine and chloramine disinfection has been demonstrated by using bacterial strains isolated from environmental sources (22, 45). The consensus hypothesis has been that resistance to sublethal concentrations of disinfectants is primarily due to interactions of the oxidants with components of bacterial membranes (e.g., sulfhydryl groups) and capsule layers. These cellular targets provide a disinfection demand and effectively decrease (with time) the concentration of disinfectant presented to a cell's membrane (8, 45, 46).
Intracellular mechanisms also contribute to disinfection resistance. Following phagocytosis, an oxidative burst is induced in a macrophage where chlorine and chloramines are formed (2, 5, 17). Accordingly, E. coli has evolved systems to resist and repair damage caused by sublethal concentrations of these oxidative compounds (17, 47). Dukan et al. (11) have demonstrated that specific heat shock (i.e., dnaK, grpE, and lon) and redox regulon (soxRS) genes are induced following sublethal exposure to chlorine. These authors proposed that resistance to chlorine disinfection is cell mediated, proceeds through a mechanism different than the mechanism used for hydrogen peroxide, and is primarily the result of injury-induced protein synthesis.
The growth conditions prior to disinfection have been shown to significantly alter the resistance of bacterial cells to chlorine and chloramines (27, 35, 44). For example, the concentration of disinfectant and the contact time required to inactivate 99% of the target bacterial population increased sixfold for chloramines and more than 200-fold for chlorine after growth in a low-nutrient medium (35, 44). In addition, induction of starvation proteins and alterations in membrane structure and composition have been shown to increase resistance to environmental stresses, including oxidative agents (26, 27). Data from the current study support these observations, as disinfection resistance increased significantly during the 29 days of starvation (Table 4).
We propose that bacterial resistance to chlorine is a biphasic process, in which the disinfectant first reacts with extrinsic components (e.g., the capsule and outer membrane) and then induces intrinsic components (i.e., the heat shock proteins and redox regulon). For example, extracellular chlorine reacts nonspecifically with components of a bacterium's capsule, outer membrane, and periplasm, reducing the concentration of disinfectant available to react with the cell's inner membrane. However, if the oxidative or disinfectant demand of the extrinsic barriers is met or overwhelmed (e.g., by increased exposure time or increased disinfectant concentration), the disinfectant diffuses to the cell's cytoplasmic membrane. Oxidative damage to this and other intracellular targets (e.g., nucleic acids) then induces the intrinsic mechanisms in an attempt to repair the resulting damage.
The ability of E. coli O157:H7 to remain physiologically active and develop resistance to chlorine has significant public health implications. Municipal water treatment facilities use surface and ground waters as source waters. Surface waters that receive nonpoint source inputs from cattle-grazing lands could provide a route of transmission to drinking water consumers. E. coli O157:H7 has been found to be widely distributed in cattle across the United States, but the prevalence was relatively low (1.6% of 11,881 fecal samples) (16). However, assuming that low prevalence rates represent an insignificant risk of infection may not be prudent, as the infective dose of the O157:H7 serotype has been estimated to be as low as 10 organisms (51). The study described here and previous studies suggest that E. coli O157:H7 could be flushed into lakes and streams from natural sources (e.g., cattle and wild bird feces) and could develop a disinfectant-resistant phenotype during transport to water treatment plants.
Drinking water treatment facilities routinely use coagulation, flocculation, sedimentation, filtration, and disinfection to remove and inactivate pathogens. However, bacteria have been shown to survive these processes and to penetrate rapid sand filters in properly operated facilities (7). In addition, our data indicate that the strain of E. coli O157:H7 which we studied can develop resistance to chlorine concentrations up to 0.5 mg/liter, a level significantly higher than the detectable disinfectant residual level mandated for drinking water distribution systems in the United States (13). Collectively, the data presented in this paper indicate that E. coli O157:H7 is a robust commensal pathogen that is capable of adapting to and surviving environmental stresses in an aquatic system.
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ACKNOWLEDGMENTS |
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This work was supported by the National Aeronautics and Space Administration (Life Sciences Program) and by U.S. Army Research Office contract G-DAAH04-96-1-0442.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, 109 Lewis Hall, Montana State University, Bozeman, MT 59717. Phone: (406) 994-5663. Fax: (406) 994-4926. E-mail: umbgm{at}montana.edu.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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