Journal ofMicrobiological Methods
Methods 26 (1996) 21-26
Journal of Microbiological
Comparison of direct PCR and PCR amplification after DNA extraction .for the detection of viable enterotoxigenic Escherichia coli in laboratory microcosms Zohreh Tamanai-Shacoori”,
Laboratoire de Microbiologic Pharmuceutique, Faculte’ de Sciences Pharmaceutiques et Biologiques, Universite’ de du Professeur LRon Bernard, 35043 Rennes, France Received
12 June 1995; accepted
Rennes I, 2, Avenue
Abstract Studies of laboratory microcosms have shown that enterotoxigenic Escherichia coli (ETEC) lose their culturability but remain viable when exposed to starvation conditions. A polymerase chain reaction (PCR) procedure was used to detect viable cells incubated in artificial seawater microcosms, either directly after boiling the water or after extracting the DNA. After 4 days of incubation, culturable bacterial counts decreased to < 1 CFU ml-’ but viable bacteria were detected for up to 6 days in all cases. After 15 days, only direct PCR remained positive and after 21 days, all results were negative. The non-culturable state of ETEC may pose health problems, and this study demonstrated the efficiency of direct PCR to detect total viable starved Ercherichia coli strains. Keywords: Enterotoxigenic
Escherichia coli; PCR; Digoxigenin
1. Introduction Enterotoxigenic Lbcherichia coli (ETEC) has been recognized as the major causative agent of bacteria diarrhea1 disease in humans and animals [ 1,2]. Immunoassays and/or bioassays of bacterial culture fluid are two methods for routine detection of ETEC [3,4]. Direct and specific detection of Escherichia coli strains carrying the elt gene coding for the heat-labile toxin (LT) by DNA colony hybridization can also be used [:5,6]. More recently, methods for detecting ETEC by polymerase chain reaction (PCR), which are a powerful in vitro tool for the
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amplification of specific DNA sequences have been developed [7-lo]. Various gram-negative bacteria are known to enter a state of non-culturability, often induced when the bacteria are exposed to adverse environmental conditions, including high salt concentration, presence of heavy metals, low nutrient level and exposure to harsh temperatures or solar radiation [II]. This has been demonstrated for several human pathogens such as Escherichia coli, Vibrio cholerae and Salmonella enteritidis [ 121, Shigella sonnei and Shigella flexneri  and Cumpylobacter jejuni . When the cells have entered the state of non-culturability, neither plating onto solid media nor inoculation into liquid media reveals the presence of viable bacteria. However, successful in vitro resuscitation and growth of
Z. Tamami-Shacoori et al. I Journal of Microbiological Methods 26 (1996) 21-26
non-culturabk cells have been demonstrated by adding nutrient media  or by varying the incubation temperature of the microcosm [ 151. It is evident that conventional culture-based bacteriological methods to detect the presence of specific waterborne pathogens may be misleading because this methodology does not permit the detection of viable but non-culturable cells. Because of the public health and ecological importance of enterotoxigenic Escherichia coli in environmental water, and the lack of knowledge regarding the viruIence of human pathogens which have entered the state of non-culturability, this study was undertaken to evaluate the use of PCR amplification for direct detection of viable enterotoxigenic Escherichia coli in laboratory microcosms.
2. Materials and methods
2.1. Bacterial strain and microcosm
An enterotoxigenic strain of Escherichia coli H10407 (LT+) , was employed in this study. For the experiments described, cells were typically grown in 100 ml of Mundell’s medium  at 37°C for 5 h (exponential phase) and 24 h (stationary phase). Cells were harvested and washed twice with artificial seawater (ASW) sterilized on a 0.22 pmpore-size filter (Millipore, USA). Cells were transferred to a sterile flask containing 500 ml of ASW solution to a final concentration of 3 x lo6 CFU ml-‘. The microcosm was maintained at room temperature (25°C) with shaking. 2.2. Total bacterial
Samples were taken at T, (inoculation time), at 2, 4, 8 and 24 h, and then on subsequent days until the bacteria were no longer culturable, for plate counts, total bacterial numbers by acridine orange direct counts (AODC). For plate counts, 0.1 ml of the water and appropriate dilutions were plated onto trypticase agar. Plates were incubated at 37°C and colonies counted after 48 h. When the viable count dropped below 10 CFU ml-‘, in order to determine CFUs at concentration of < 1 CFU ml-’ (at which concentration the cells were considered to be in the
non-culturable state), 10 ml of the microcosm was removed and filtered through a 0.22 pm-pore-size sterile filter, placed on the solid medium, and observed for growth at 37°C. Total bacterial numbers were estimated by staining formalin-fixed cells with a 0.1% acridine orange solution as previously described by Oliver 1181. 2.3. DNA extraction Enterotoxigenic Escherichia coli H10407 DNA to be used as a PCR template was extracted from ASW culture by two methods: 2.3.1. GUSCN method Total DNA was extracted according to the method of Boom et al. [ 191. A 1.5 ml sample of water was centrifuged at 12 000 g for 20 min (or 15 000 g for 5 min) in an Eppendorf tube (Eppendorf type 3810). The pellet was resuspended in 940 ,ul of a silica solution (40 ~1 of silica added to 900 ~1 of lysis buffer consisting of 120 g guanidinium thiocyanate (GUSCN), dissolved in 100 ml of 0.1 M Tris HCl, .pH 6.4, and added to 22 ml of 0.2 M EDTA, pH 8, and 2.6 g Triton X100>, and incubated for 10 min at room temperature. After centrifugation at 12 000 g for 10 min, the supematant was drawn off and discarded. Each pellet was washed twice with washing buffer (120 g GUSCN in 100 ml of 0.1 M Tris HCI, pH 6.4), twice with 70% ethanol (v/v), and once with acetone. The pellet was then dried at 56°C for 10 min. The DNA was eluted from the pellet with TE buffer (0.01 M Tris HCl, 0.001 M EDTA), incubated at 56°C for 10 min, and centrifuged for 2 min at 12 000 g. All the supematant (10 ~1) containing DNA was used for PCR analysis. 2.3.2. Whole cell preparations Ten microlitres of ASW culture in an Eppendorf tube was placed in a 100°C water bath for 10 min and then used for direct PCR amplification. 2.4. PCR primers Primers were based on the nucleotide sequence of a 322 bp region of the B subunit of the Escherichia coli elt gene. The two different 19 and 20 base primers , LT, [5’-CCATACTGATTGCCGC-
Z. Tamanai-Shacooriet al. I Journal of MicrobiologicalMethods26 (1996) 21-26
AAT-3’1 and LT, [5’-TCTCTATGTGCATACGGAGC-3’1 were synthesized with an automated DNA synthesizer (Applied Biosystems Inc., Foster City, CA) by the phospharamidite method. 2.5. DNA amplification and synthesis of probe by PCR
ualized by reaction with NitroBlue Tetrazolium solution (75 mg ml-’ in 70% dimethylformamide) and 5-bromo4-chloro-3-indolylphosphate solution at room tem(40 mg ml-’ in dimethylformamide) perature in the dark for periods ranging from 20 min to 1 day.
PCR amplification was performed in a DNA thermal cycler (Hybaid, Teddington, UK), in a final volume of 100 ,ul containing 25 ng of template DNA or 10 ,ul of whole cell preparation of each sample, 1 X reaction buffer (0.02 M (NH,)*SO,, 0.08 M Tris HCl pH 9, 0.0025 M MgCl,), 4 U of Thermus aqclaticus polymerase (Beckman Instruments, USA), 0.2 mM each of th.e dNTPs, and 10 ,uM of each primer. The solutions were subjected to 30 cycles of amplification with a 30 s denaturation at 95”C, 30 s re-annealing at 55°C and 1 min extension at 72°C. After the last cycle., samples were kept at 72°C for 10 min to complete elongation. The same procedure was used to label D:NA with digoxigenin  except that dTTP was added at 26 PM with 13 PM of Dig-dUTP (Boehringer Mannheim, Germany).
3. Results and discussion Escherichia coli H10407 populations were monitored for total and viable cells during starvation in ASW at 25°C. Fig. 1 shows the survival curves for this organism in this type of microcosm inoculated with 3 X lo6 CFU ml-’ of exponential and stationary phase cultures. As can be seen, we observed no decrease in plate counts for stationary phase inocula even after 3 weeks of starvation. For the exponential phase, plate counts declined gradually, typically from lo6 CFU ml-’ to < 1 CFU ml-’ (non-culturable state) after 4 days and < 0.1 CFU ml-’ after 8 days
2.6. Detection of amplified DNA An aliquot (10 r~l) from the PCR product was analyzed by gel electrophoresis in 2% agarose gels (Seakem; FMC Bioproducts, Rockland, ME) in 40 mM Tris-acetate (PI-I 8) and 1 mM Na,EDTA buffer for 1.5 h at 80 V. The gels were stained in ethidium bromide (0.5 ,ug ml-‘, Sigma) for 15 min and photographed on a UV transilluminator. Molecular weight standard VIII DNA ladder (Boehringer Mannheim, Germany) was included. For Southern blot analysis , the electrophoresed DNA was transferred onto a Nylon membrane (Hybond N., Amersham, UK). The membranes were pre-hybridized for at least 1 h at 42°C and hybridized with 25 ng ml-’ of heat-denaturated PCR-labeled probe at 42°C overnight in ,accordance with the manufacturer’s recommendations (Boehringer Mannheim, Germany). Subsequently, they were washed twice in 2 X SSC-0.1% SDS at room temperature for 5 min, and then twice again in 0.1 X SSC-0.1% SDS at 55°C for 10 min. ‘The hybridization reactions with the alkaline phosphatase-conjugated probe were vis-
starvation dme(days) Fig. 1. Comparison of survival response of stationary and exponential phase cells of Escherichia coli H10407 starved in ASW at 25°C. Acridine orange direct counts for stationary or exponential phase (A), plate counts for stationary phase (m), and plate counts for exponential phase (0).
et al. I Journal of Microbiological
of starvation. At that stage, the total cell numbers of all populations determined by AODC, remained at a nearly constant cell density of 2 X IO6 to 3 X lo6 cells ml - I, showing no changes during the entire starvation period. The Escherichia coZi H10407 population entered a state of non-culturability by day 4 or 5 of incubation in ASW. Between day 4 and day 8, non-culturability appeared to be reversible by transfer from the oligotrophic environment to a richer medium such as trypticase agar, for an extended incubation time ranging from 48 to 72 h. From day 8 to the end of the experiment (day 21), the ability to form colonies upon plating on a solid medium disappeared irreversibly (data not shown). According to Postgate , bacteria may lose their ability to form colonies and yet remain functional as individual cells. This may pose health problems beyond this period, because results of animal studies have demonstrated that non-culturable cells continue to harbor a potential for virulence [ 131. Vibrio cholerae has been shown to cause diarrhea1 symptoms in volunteer studies by reversion of non-culturable bacteria to a culturable form . The possibility of resuscitating such cells could provide a mechanism whereby non-culturable cells, in a metabolically dormant condition, could revert to a state of increased metabolism when environmental conditions are more conducive [ 151. Our results suggest that the survival of Escherichia coli H10407 in seawater depends on the
Methods 26 (1996) 21-26
age of the culture during prior growth. Gauthier et al.  studied the influence of prior growth conditions of Escherichia coli using experimental microcosms. They concluded that the sensitivity of all cells was highest when they were harvested during the early exponential phase of growth, before inoculation in nutrient-free seawater. Oliver et al.  have reported that, of 10 factors studied which might affect the non-culturable response in Vibrio vulnijicus, only the physiological age of the culture was found to significantly affect the rate at which cells became non-culturable. We also observed, as they did, a progressive diminution in colony diameter and a morphological change from rods to predominantly cocci on the agar medium when the bacteria entered a non-culturable state. Whole cell preparations and/or total DNA from Escherichia coli H10407 in the non-culturable state made at day 4, 6, 8, 15 and 21 after inoculation were subjected to PCR amplification. The profiles of the amplification products were analyzed by agarose gel electrophoresis (Fig. 2A). Correct amplification was confirmed by transfer of the DNA to Nylon membrane, and testing for hybridization with an LTspecific probe (Fig. 2B). As shown in these figures, the use of total extracted DNA as a template in amplification of the eZt gene allows detection of enterotoxigenic Escherichia coli even after 6 days of starvation. The whole cell preparation directly amplified, without primary extraction (direct PCR),
Fig. 2. Agarose gel electrophoresis and Southern hybridization of PCR products. (A) PCR products separated on a 2% agarose gel; (B) PCR products blotted and hybridized with LT-specific probe. Lanes: 1, DNA molecular weight marker VIII (Boehringer); 2, 4, 6, 8, 10, 12, Escherichia coli H10407 detected in ASW by PCR after DNA extraction; 3, 5, 7, 9, 11, 13, Escherichia coli H10407 detected in ASW by direct PCR, respectively, after 2 (positive control), 4, 6, 8, 15 and 21 days of starvation.
Z. Tamanni-Shacoori et al. I Journal of Microbiological Methods 26 (1996) 21-26
gave positive results even after 15 days of incubation in ASW. At day 2’1 of starvation results with both techniques were negative. PCR amplification results obtained on day 2 of starvation were used as positive controls. Positive PCR results, obtained from ASW surcharged with a variable number of LT+ bacteria by two methods, eliminated the presence of inhibitory substances. In the present study, the persistence of enterotoxigenic Escherichia coli in ASW was observed by PCR on both extracted DNA and whole cell preparations. Direct PCR seems to be a more sensitive method of cnterotoxigenic Escherichia coli detection under starvation conditions. The difference of sensitivity could be explained by the low percentage of starved bacteria collected by centrifugation  in plasmid extraction for PCR amplification. It is worth noting that the yields of amplified DNA by direct PCR were slightly lower than those obtained using extracted DNA. No difference in yield of amplified DNA was detected when using distilled water as laboratory microcosms (unpublished results). This observation may be explained by the inhibitory effect of salinity on PCR detection. In this study, the positive results from PCR amplification after 4 days of incubation in ASW may arise from the presence of either viable but nonculturable ETEC, or from intact nucleic acids from dead cells in samples, prior to degradation as illustrated by Josephson et al. . These authors concluded that despite the rapid degradation of nucleic acids in environmental samples, there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. In the environment, DNA thus amenable to PCR amplification can subsequently act as a transforming agent. Islam et al.  have detected non-culturable but viable Shigella dysenteriae 1 in a laboratory microcosm by PCR amplification of extracted DNA. The maintenance of inFectivity is at present contentious because studies are controversial. The non-culturable stage appears to be a strategy for survival for bacteria exposed to starvation conditions such as high salinity, low nutrient medium, sunlight, etc. It is important to detect such cells, especially ETEC strains which can produce LT and/or ST enterotoxin and be implicated in outbreaks of diarrheal diseases in humans and young animals.
Acknowledgments We gratefully acknowledge D. Ni Eidhin’s and G. Bouer’s critical review of the manuscript. We also thank Brigitte Bardot for typing the manuscript.
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