APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1993, p. 536-5400099-2240/93/020536-05$02.00/0

Use of the Polymerase Chain Reaction and Fluorescent-Antibody Methods for Detecting Viable but NonculturableShigella dysenteriae Type 1 in Laboratory Microcosms

M. S. ISLAM,`* M. K. HASAN,1 M. A. MIAH,1 G. C. SUR,' A. FELSENSTEIN,'M. VENKATESAN,2 R. B. SACK,' AND M. J. ALBERT'

International Centre for Diarrhoeal Disease Research, P. 0. Box 128, Dhaka 1000, Bangladesh,and Walter Reed Anny Institute of Research, Washington, D. C. 20307-51002

Received 5 August 1992/Accepted 11 November 1992

Epidemiological studies of shigellosis in Bangladesh have demonstrated that surface-water sources can act asfoci of infection. Studies of laboratory microcosms have shown that shigellae become nonculturable but remainviable when exposed to environmental samples of water. The present study was carried out to detect viable butnonculturable Shigella dysenteriae 1 from laboratory microcosms by the polymerase chain reaction and thefluorescent-antibody techniques. S. dysenteriae 1 was inoculated into laboratory microcosms consisting of watersamples collected from ponds, lakes, rivers, and drains in Bangladesh. The survival of S. dysenteriae inmicrocosms was assessed by viable counting on MacConkey agar. After 2 to 3 weeks, S. dysenteriae 1 becamenonculturable but remained viable. After 6 weeks, this nonculturable but viable S. dysenteriae 1 was detected byboth the polymerase chain reaction and the fluorescent-antibody methods. The viable but nonculturable state ofS. dysenteriae 1 demonstrated in this study may be important for understanding the epidemiology of shigellosis.

Shigellosis is a major health problem, resulting in highrates of morbidity and mortality in Bangladesh (10, 18); it isresponsible for the highest mortality rate of all of thediarrheal illnesses among hospitalized patients (19). Epide-miological studies of shigellosis in Bangladesh have shownthat surface water sources, e.g., ponds, lakes, wells, andrivers, can act as sources of infection (19). In the UnitedStates, outbreaks of shigellosis have also been attributed toswimming in contaminated water (27).

Colwell et al. (4) observed that when Shigella sonnei wasexposed to laboratory microcosms consisting of ChesapeakeBay water, it became nonculturable but remained viableafter 21 days. They detected the organisms in this state byfluorescent-antibody methods. Thus, the potential publichealth hazard presented by such Shigella species existing inthe nonculturable state may be significant, because it hasbeen found that human volunteers developed clinical symp-toms after ingestion of nonculturable cells of Vibrio choleraewhich were also subsequently isolated in culturable formfrom the stools of volunteers (5). Moreover, it has recentlybeen shown that nonculturable bacteria can be resuscitatedto a culturable state (24). However, one difficulty in eluci-dating the potential hazard of viable but nonculturablepathogenic bacteria is the inability to detect such cells in thenatural environment by employing routine bacteriologicalmethods. Furthermore, any detection method that is em-ployed must be capable of detecting low numbers of shigel-lae against a large background of other cells and of organicmaterial which may be present in the sample. One methodfor detecting such nonculturable bacteria is the polymerasechain reaction (PCR). This technique allows a specific seg-

ment of the DNA to be amplified by a factor of 106 or more

within hours (29), thus potentially permitting the detection ofcells that are present in low numbers.PCR methodology depends only on the presence of target

* Corresponding author.

DNA and not of culturable cells; thus, PCR is potentiallyable to detect the presence of viable but nonculturable cells.However, virtually no data on the detection of nonculturablebut viable shigellae from environmental samples exist. Long-term survival of low numbers of nonculturable yet viableshigellae could have implications for the transmission ofdisease. Such shigellae can be detected by techniques whichdo not rely on culturing, such as the PCR and the fluores-cent-antibody techniques. Therefore, the present study wasundertaken to investigate nonculturable but viable Shigelladysenteriae by both the PCR and the fluorescent-antibodymethods in laboratory microcosms containing water col-lected from various surface-water sources of Bangladesh.

MATERIALS AND METHODS

Survival experiments. All of the experiments in this studyare similar in design, including the addition of a number oforganisms to a measured volume of water and the subse-quent monitoring of the viable numbers over a defined periodof time.

Bacterial strain. One strain of S. dysenteriae type 1 (strain14444) obtained from the Clinical Microbiology Laboratoryof the International Centre for Diarrhoeal Disease Research,Dhaka, Bangladesh (ICDDRB) was used in this study. Thisstrain was reconfirmed by cultural, morphological, biochem-ical, and serological tests according to standard procedures(17). In brief, when this strain was subcultured onto Mac-Conkey agar (MA) and Salmonella-Shigella agar media, itproduced non-lactose-fermenting colonies. Non-lactose-fer-menting colonies were inoculated into Kligler iron agar,motility indole urea, and Simmons citrate agar media. Thereactions in Kligler iron agar medium were acid butt andalkaline slant without production of gas and H2S. This strainwas nonmotile and was negative for urea and indole inmotility indole urea medium. It could not utilize citrate as asource of carbon. All of these biochemical reactions aretypical of Shigella species. By slide agglutination, it was

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found that this strain agglutinated with S. dysenteriae type 1antiserum, purchased from Wellcome Diagnostics (Dartford,England).

Preparation of inoculum. The strain was first inoculatedonto MA (Difco) and incubated at 37°C for 24 h. A loopful ofthe resulting growth was resuspended in test tubes contain-ing sterile samples (10 ml each) of pond, lake, river, anddrain water samples. Water from these sources was steril-ized by autoclaving at 121°C for 15 min at a pressure of 15 lb[ca. 6800 g]. These autoclaved water samples were alsoprechecked for the presence of amplifiable Shigella DNA byPCR and were found to be negative.) The transmittance ofthe suspension at A585 was determined with a spectropho-tometer (Coleman Junior IIA, model 6/20A; Perkin-ElmerCorp.) and the concentration of the cell suspension wasadjusted to the desired level. Then, the numbers of cells permilliliter were assessed by viable counts by the drop platetechnique.

Preparation and inoculation of suspending fluid and storageof flasks. In 500-ml conical glass flasks (Pyrex), 100 ml eachof autoclaved pond (Institute of Public Health, Mohakhali,Dhaka, Bangladesh), lake (Dhanmandi Lake, Dhaka), TongiRiver, and drain water samples (Paramedical Institute,Dhaka) was taken. Then, a measured inoculum of about 105S. dysentenae 1 organisms per ml was added and mixed. Theflasks were then stored at room temperature (25°C).

Sampling timetable and counting procedure. Culturablecells were counted at 0, 1, 2, 4, 8, and 24 h and then atdifferent days until the bacteria were no longer culturable.The counting of any suspension was discontinued afterfailure to recover S. dysenteniae type 1 from two consecutivesamplings. For each time, a 0.1-ml sample from each flaskwas taken and 10-fold dilutions were prepared in phosphate-buffered saline (PBS); then, 25 ,ul from different dilutionswas plated on MA according to the drop plate technique (8).Bacterial counts were expressed as CFU per milliliter. Whenno culturable cells were found after two consecutive sam-plings from a 10-1 dilution, 0.1 ml of water was taken fromthe microcosm and plated onto MA. When the number ofculturable cells became less than 10 CFU per ml, 1.0 ml ofwater was removed from each microcosm and centrifuged at15,000 x g for 5 min in a Microfuge (model 5415C; Eppen-dorf, Gosheim, Germany). The pellet was plated on MA andincubated at 37°C for 72 h. This was done to increase thedetection limit to 1.0 CFU per ml.DNA preparation. One milliliter of water was removed

from each microcosm and centrifuged at 15,000 x g for 5 minin an Eppendorf Microfuge (model 5415C). Each pellet wasresuspended in 50 pl of a solution containing 50 mM Tris-HCl (pH 8.0), 20% sucrose, 50 mM EDTA, and 400 p,g oflysozyme (Sigma, St. Louis, Mo.) per ml and incubated at37°C for 30 min. Next, 150 ,ul of a solution containing 50 mMNaCl, 1% sodium dodecyl sulfate (SDS), and 500 p,g ofproteinase K (Bethesda Research Laboratories, Gaithers-burg, Md.) per ml was added, and the mixture was incubatedat 50°C for 60 min. The DNA was precipitated with 2volumes of absolute ethanol in the presence of 0.3 M sodiumacetate (pH 5.2) at -70°C, harvested by centrifugation at15,000 x g for 15 min, and resuspended in 100 pl of asolution containing 10 mM Tris-HCl and 1 mM EDTA (pH8.0).DNA amplification. DNA was amplified by PCR with 1.0 RI

of the extracted DNA with 130 ng each of two primers(primer H8 [5'-GTTCCITGACCGCCTVTCCGATAC-3']and primer H15 [5'-GCCGGTCAGCCACCCTC-3'] [7]) and1.25 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk,

Conn.) per 25 ,ul of reaction mixture on a Thermal Cycler(Perkin-Elmer Cetus). The PCR reaction was done for 35cycles of 1 min each at 94°C (for denaturation), 1.5 min eachat 60°C (for annealing of primers to single-stranded DNA),and 0.25 to 1 min each at 72°C (for DNA polymerase-mediated extension) according to the procedure describedby Escheverria et al. (6). Amplified DNA was separatedelectrophoretically on a 0.8% agarose gel and transferred toa nylon membrane (Sigma) by capillary action (23).DNA hybridization. Nylon membranes containing DNA

were prehybridized in prewarmed hybridization buffer (5xSSC [lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate],1.0% SDS, 0.5% bovine serum albumin) for 10 min at 37°C.The filters were hybridized in the same buffer containing 2.7,ul of a 5 ,uM stock solution of the alkaline phosphatase-labeled ipaH probe per 55-cm2 filter at 37°C for 30 min. Theprobe is basically the fragment that is amplified with primersH8 and H15. The membranes were then washed once for 10min in 1% SDS-1 x SSC at 37°C, once for 10 min in 1%Triton x-100-lx SSC at 37°C, and once for 10 min in lxSSC at 45°C. Hybridization was carried out according to theprocedure described by Joblonski et al. (15). The primersand probe were provided by D. J. Kopecko of the WalterReed Army Institute of Research, Washington, D.C.The membranes were then transferred to a solution of 7.5

ml of alkaline phosphatase buffer (100 mM Tris-HCl, 100mM NaCl, 50 mM MgCl2, 0.1 mM ZnCl2, and 0.02% sodiumazide; pH 8.5) containing 33 p.I of nitroblue tetrazolium(Sigma) solution (75 mg/ml in 70% dimethylformamide) and25 p.l of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) solu-tion (50 mg/ml in dimethylformamide). The color reactionmixture was incubated at room temperature for 4 h in thedark. The reaction was terminated by washing the filterswith distilled water.

Fluorescent microscopy. One milliliter of water was re-moved from each microcosm and centrifuged at 15,000 x grpm for 5 min. Then, the pellet was resuspended in 25 ,ul ofwater taken from the same microcosm. A 5-pA volume of thissuspension was placed on polytetrafluoroethylene-coatedwell glass slides. The slides were air dried and flame fixed.Then, 1 drop (20 pA) of 1:10 S. dysenteriae type 1 polyclonalantiserum (Wellcome) was added to the wells, and themixture was incubated at 37°C for 30 min in a moistincubation chamber. After incubation, the slides were rinsedin distilled water, gently blotted, and air dried. Fluoresceinisothiocyanate-conjugated anti-rabbit goat serum (20 pA)diluted in PBS (1:80) was added to the wells and incubatedfor 30 min at 37°C. The slides were then washed, dried, andmounted under a coverslip with buffered glycerol (pH 8.9).Finally, the slides were examined under an epifluorescencemicroscope (model BH-2; Olympus). S. dysenteriae 1 14444(culturable) was used as a positive control. We also includeddifferent Shigella species, e.g., S. flexneri, S. boydii, S.sonnei and other enteric bacteria, such as Escherichia coli,Klebsiella species, and Enterobacter species, which haveshown negative reactions with S. dysentenae 1 antiserum.

Viability testing. Viability testing of nonculturable S. dys-enteniae 1 was carried out according to the proceduredescribed by Kogure et al. (20). In brief, nonculturable cellsare added to the solution containing yeast extract (0.025%)as a nutrient supplement and nalidixic acid (0.002%) as anantibiotic which inhibits cross wall formation upon celldivision by inhibiting DNA gyrase and terminating DNApolymerization (35). Hence, viable cells will start growingbut will not be able to divide. Upon staining with acridine

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7

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0 4a;,0-J

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FIG. 1. Culturability of S. dysentenae 1 in different types ofwater in MA medium.

orange (0.01%) and observation under UV light, viable cellsappear greater than their normal length.

RESULTSFigure 1 shows the culturability of S. dysenteriae 1 in

different types of water as revealed by viable counting onMA. On MA plates, the counts of drain water were alwayshigher than those of other sources from day 1. The secondhighest count was for lake water. Most of the time, thelowest count was observed for river water. For pond water,most of the time, the count was higher than that for riverwater but lower than those for lake and drain water samples.The longest survival observed was for drain water (up to 19days), and the shortest was for river water (up to 13 days).The DNAs isolated from the nonculturable state of S.

dysenteriae 1 14444 obtained from microcosms after 3 weeksof survival study were subjected to PCR, and the productswere analyzed by agarose gel electrophoresis (Fig. 2A). TheDNAs were transferred to nylon membranes and werehybridized against the ipaH probe. The four water samplescollected from drain, pond, lake, and river microcosmscontaining nonculturable S. dysentenae 1 generated theexpected 700-bp fragment of theH locus which hybridized tothe ipaH probe (Fig. 2B).The water samples from the microcosms were examined

by fluorescence microscopy 3 weeks after the end of thesurvival study, when the bacteria were nonculturable. It wasfound that S. dysenteriae 1 was still present in the micro-cosms (Fig. 3A). Figure 3B shows the culturable S. dysen-teriae 1 14444 which was used as a positive control.Three weeks after the end of the survival study, viable

cells of S. dysenteriae 1 were observed under UV lightaccording to the procedures described by Kogure et al. (20).

DISCUSSIONIn the present study, S. dysenteriae 1 was shown to enter

a viable but nonculturable state 2 to 3 weeks after inocula-tion into laboratory microcosms. This state, which may be asurvival stage, is an example of the so-called death phase ofthe growth stages of a microorganism. The death phase was

FIG. 2. (A) Specific amplification of invasion plasmid antigen Hlocus (ipaH) DNA by the PCR. Amplified products of each reactionwere analyzed by electrophoresis through a 0.8% agarose gel.Lanes: 1, S. flexneri M9OT used as positive control (culturable); 2,negative control without template; 3 through 6, nonculturable S.dysenteriae 1 detected from drain, pond, lake, and river watermicrocosms, respectively, 6 weeks after inoculation; 7, HaeIIIdigest of X174 replicative-form DNA. (B) Hybridization of Southernblot of panel 2A with alkaline phosphatase-labeled ipaH probe.Lanes: 1, S. flexneri M9OT used as positive control (culturable); 2,negative control without template; 3 through 6, nonculturable S.dysenteriae 1 detected from drain, pond, lake, and river watermicrocosms, respectively, 6 weeks after inoculation.

reevaluated recently after methods to assess the viability ofthese nonculturable cells, including direct viable counting(20) and microautoradiography (9), had been developed. Thenonculturable stage in the life cycle of a bacterium appearsto be a strategy for survival when the organism is exposed toconditions that are less than optimal for cell growth anddivision.

Studies have shown that V. cholerae, Campylobacterspecies, and shigellae (nonculturable) fail to grow in conven-tional cultural media but remain viable when exposed tolaboratory microcosms (4, 14, 16). It is therefore importantthat methods to detect such cells be developed. Moreover, atechnique to detect small numbers of cells is essential, sincemany bacteria are present in the natural environment only atlow cell densities (1, 11-13). Such detection is especiallyimportant for bacteria such as shigellae which can producedisease with as few as 10 organisms (22). The PCR techniqueseems ideally suited for this goal, since this method poten-tially allows amplification of the DNA obtained from as fewas one cell (31). It is therefore essential that the portion ofDNA to be amplified by this technique be unique to thespecies of bacterium under investigation. The invasion plas-mid antigen gene (ipaH) of shigellae has previously beencloned (2) and sequenced (7), and this gene has been shownto be unique to shigellae and enteroinvasive E. coli (34).

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FIG. 3. Fluorescence photomicrography of S. dysenteriae 1. (A)Nonculturable S. dysenteriae 1 14444 (obtained from microcosms;magnification, x 1,000); (B) culturable S. dysenteriae 1 14444 used as

a positive control (magnification, x 1,000).

Studies to detect lower numbers of shigellae from clinicaland environmental samples with ial probes have been con-

ducted elsewhere (6). DNA probes against the large invasiveplasmid of enteroinvasive bacteria have been targeted (30,32, 36). However, the plasmid is known to be unstable afterlong storage in culture collection (3), and the plasmid can beeasily lost after overnight culture under nonselective or

competitive conditions (21). Use of the ipaH probe facili-tated overcoming this problem, because this gene is present

in more than one copy on both the invasion plasmid and thechromosome (33).There is circumstantial evidence suggesting that S. dysen-

teriae 1 survives for extended periods in natural aquaticenvironments after deposition by humans attributable toindiscriminate defecation in countries like Bangladesh, be-cause water sources have been implicated in the outbreaksof shigellosis (25). This has led us to explore the viable butnonculturable form of S. dysenteriae, since it has beenshown to occur in V. cholerae, Salmonella ententidis,enteropathogenic E. coli, and other waterborne pathogens(4, 28, 37).

In the present study, the persistence of S. dysenteriaecells in laboratory microcosms has been observed by fluo-rescence microscopy for up to 6 weeks, despite the fact thatat 6 weeks the cells cannot be cultured with solid medium.The results with direct viable counting showed that thesenonculturable cells retained viability, as shown for V. chol-erae, Campylobacter species, and related enteric water-borne pathogens (4, 26). Thus, nonculturability of S. dysen-teniae 1 on growth medium cannot be equated withnonviability.By extrapolating from these findings to the natural envi-

ronment, it is concluded that the present methods of detect-ing viability by conventional cultural techniques are inade-quate. This hypothesis is corroborated by outbreaks ofshigellosis in which no organism can be isolated from thesuspected transmission vehicles by conventional culturaltechniques (25). However, the PCR and the fluorescent-antibody methods provide the opportunity of detecting theseviable but nonculturable S. dysenteriae cells even if they arepresent in very low numbers.The viable but nonculturable stage reported here for S.

dysenteriae 1 is significant for understanding the epidemiol-ogy of shigellosis. If these viable but nonculturable cells areingested by humans, they may cause disease, as has beenshown for V. cholerae in volunteer studies in which noncul-turable bacteria caused disease in volunteers by reversion toculturable form (5). People in developing countries such asBangladesh extensively use waters from various surface-water sources, e.g., ponds, lakes, and rivers, etc. for variouspurposes, including drinking. Therefore, the nonculturablestate of shigellae may pose health problems. The results ofthis study, therefore, demonstrated the significance of thesurvival of viable but nonculturable S. dysenteriae 1 insurface waters, which is important from a public-health pointof view.

ACKNOWLEDGMENTSThis research was supported by the Swiss Development Corpo-

ration and the ICDDRB. The ICDDRB is supported by countriesand agencies which share its concern for the health problems ofdeveloping countries. Current donors include the aid agencies of thegovernments of Australia, Bangladesh, Belgium, Canada, Denmark,France, Japan, the Netherlands, Norway, Saudi Arabia, Sweden,Switzerland, the United Kingdom, and the United States; interna-tional organizations including the United Nations DevelopmentProgramme, the United Nations Children's Fund, and the WorldHealth Organization; and private foundations including the FordFoundation and the Sasakawa Foundation.We are grateful to Dennis J. Kopecko of the Walter Reed Army

Institute of Research, Washington, D.C., for providing the primersand probes and to Manzurul Haque of the ICDDRB for his secre-tarial assistance.

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