Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholerae

M. Kamruzzaman; S. M. Nashir Udden; D. Ewen Cameron; Stephen B. Calderwood; G. Balakrish Nair; John J. Mekalanos; Shah M. Faruque

doi:10.1073/pnas.0913404107

New Research In

Contributed by G. Balakrish Nair, November 24, 2009 (sent for review October 20, 2009)

Abstract

The factors that enhance the waterborne spread of bacterial epidemics and sustain the pathogens in nature are unclear. The epidemic diarrheal disease cholera caused by Vibrio cholerae spreads through water contaminated with the pathogen. However, the bacteria exist in water mostly as clumps of cells, which resist cultivation by standard techniques but revive into fully virulent form in the intestinal milieu. These conditionally viable environmental cells (CVEC), alternatively called viable but nonculturable cells, presumably play a crucial role in cholera epidemiology. However, the precise mechanism causing the transition of V. cholerae to the CVEC form and this form's significance in the biology of the pathogen are unknown. Here we show that this process involves biofilm formation that is dependent on quorum sensing, a regulatory response that is controlled by cell density. V. cholerae strains carrying mutations in genes required for quorum sensing and biofilm formation displayed altered CVEC formation in environmental water following intestinal infections. Analysis of naturally occurring V. cholerae CVEC showed that organisms that adopt this quiescent physiological state typically exist as clumps of cells that comprise a single clone closely related to isolates causing the most recent local cholera epidemic. These results support a model of cholera transmission in which in vivo-formed biofilms convert to CVEC upon the introduction of cholera stools into environmental water. Our data further suggest that a temporary loss of quorum sensing due to dilution of extracellular autoinducers confers a selective advantage to communities of V. cholerae by blocking quorum-mediated regulatory responses that would break down biofilms and thus interfere with CVEC formation.

Bacterial gene regulation in response to cell density, known as “quorum sensing,” is a regulatory response thought to occur in bacterial communities through the sensing of extracellular signal molecules called autoinducers that are produced by members of the community (1, 2). Quorum-sensing systems (Fig. S1) such as those described in Vibrio cholerae, the causative agent of the epidemic diarrheal disease cholera, have been shown to regulate certain phenotypes, including biofilm formation and virulence (1 ⇓⇓–4). As a waterborne pathogen, V. cholerae is known to transit between the host intestinal milieu and a hypotonic aquatic environment during spreading epidemics of cholera (5), and both biofilm formation and quorum sensing have been proposed to influence transmission of V. cholerae (2 ⇓–4, 6). Although phage predation (7 ⇓–9), along with other regulatory changes (10, 11) that have been proposed to occur in vivo and in vitro, might also influence transmissibility, the genetic mechanisms that promote survival of V. cholerae in the natural aquatic environments (and thus support waterborne disease) remain unknown.

Although cholera is a waterborne disease (12 ⇓–14), the concentration of pathogenic V. cholerae, which is usually detected in surface water by standard culture, is far less than that required to induce infection and cause clinical disease in volunteers challenged with V. cholerae (15). This discrepancy between the required infectious dose and the apparent concentration of V. cholerae in water fostering an epidemic led to studies that established that pathogenic V. cholerae exist in the water as clumps of metabolically impeded cells (6). These environmental survival forms of bacteria, referred to as conditionally viable environmental cells (CVEC), a term that is perhaps synonymous with viable but nonculturable (VBNC) cells (16), resist cultivation by standard methods. CVEC can be cultured using a modified enrichment technique (17) but can revive into fully culturable and virulent form when inoculated into the ileal loops of adult rabbits (6).

V. cholerae uses quorum sensing to monitor its own cell density and to form surface-associated communities of bacterial cells known as biofilms (2, 3). The life cycle of V. cholerae in the host also includes a phase in which the bacterium colonizes the intestinal surface in a thick mat of cells resembling a biofilm (18). In this study, we show that the genetic regulators of quorum sensing and biofilm formation also affect the development of CVEC. Furthermore, we explore a possible mechanism in the metabolic transition of V. cholerae to the CVEC state and explain the significance of this survival form in the environmental persistence and epidemic spread of pathogenic V. cholerae.

Results

CVEC Are Derived from Quorum-Regulated Biofilms.

V. cholerae quorum-sensing systems have been found to influence biofilm formation in vitro as well as intestinal colonization in vivo (2 ⇓–4). In this process, the information from the sensory circuits converges to the response regulator protein LuxO, which negatively controls the transcriptional regulator HapR, which in turn controls certain genes involved in the formation or dissolution of biofilms and in regulating virulence genes involved in intestinal colonization (Fig. S1). Because CVEC exist naturally as clumps of cells that resemble biofilm communities (6), we sought to determine if development of CVEC and biofilms are controlled by the same genetic regulators. We analyzed the El Tor biotype strain C6706 and its derivatives that carry defined mutations for their ability to develop biofilms and CVEC under different conditions (Figs. 1–3 and Fig. S2).

Fig. 1.

Biofilm formation by strain C6706 and its mutants carrying insertions in different genes under laboratory conditions (A) and in the ileal loops of adult rabbits (B). Formation of clumps of cells under in vivo conditions is down-regulated in the luxO mutant but up-regulated in the hapR mutant, similar to the pattern of biofilm formation in vitro. The cells were rendered fluorescent due to expression of GFP from the TnFGL3 inserts in the mutant strains or from a GFP-expressing plasmid introduced into the wild-type strain C6706.

Fig. 3.

Formation of CVEC by V. cholerae excreted in the stools of cholera patients. Mean count of V. cholerae observed from planktonic and clumped cell fractions of seven different cholera stools when inoculated in filter-sterilized environmental water samples and incubated at room temperature. The presence of cells could be detected by the AST culture method up to 45 days postinoculation with clumped cells. However, planktonic cells could not be recovered after ∼9 days.

Biofilm formation by V. cholerae cells in vivo was tested by inoculating the strains in ileal loops of adult rabbits, a well-established animal model for cholera-like disease (19). After 16 h of inoculation, the ileal loop fluid was examined, and two distinct populations of cells—free-swimming planktonic cells and biofilm-like clumped cells—were found. In agreement with the results of in vitro assays (Fig. S2), these in vivo experiments showed that an isogenic strain carrying a mutation in hapR produced more robust biofilm-like clumps than the wild-type strain, whereas the luxO mutant produced very little biofilm (Fig. 1 A and B). Additionally, mutants carrying insertions in genes encoding enzymes required for Vibrio exopolysaccharide synthesis (EPS) were also very poor biofilm producers. In general, mutants that produced more robust biofilms under in vitro conditions also produced more compact and larger clumps of cells in vivo within the intestine and vice versa. By a semiquantitative dot-blot hybridization analysis of total V. cholerae DNA isolated from single clumps of cells, we estimated that an average-sized clump in the rabbit intestinal fluid could hold ∼4.2 × 10⁸ V. cholerae cells. Thus, one such clump could constitute a human infectious dose on the basis of volunteer studies (15).

To determine if CVEC formation is induced following intestinal infection, V. cholerae present in the rabbit ileal loop fluids were introduced into samples of filter-sterilized environmental water and monitored for the formation of CVEC-like cells using a previously described assay (6, 17). By definition, CVEC are naturally occurring, environmental V. cholerae cells that are metabolically defective and are thus unable to form colonies on certain antibiotic-containing nutrient agar plates despite carrying the genes encoding resistance to the antibiotic. However, when these same CVEC are preincubated in antibiotic-free nutrient broth for at least 5 h, they become culturable on the antibiotic-containing plates in an approach called the antibiotic selection technique (AST) procedure (17). In the present study, this differential antibiotic resistance phenotype was conveniently used to define CVEC-like cells that formed under laboratory conditions, which, for convenience, will be referred to as CVEC-L here. Specifically, metabolically normal cells carrying the transposon TnFGL3, which was used to construct the insertion mutants (20), retained the ability to express the TnFGL3-encoded resistance to kanamycin, whereas laboratory-induced CVEC-L became sensitive to this antibiotic. However, resistance to streptomycin due to chromosomal mutation was expressed by both CVEC-L and normal cells, enabling us to accurately measure CVEC-L formation efficiency (see Materials and Methods).

Strain C6706 and its derivatives that were proficient in biofilm formation produced CVEC-L in our assays (Fig. 2A). To analyze the propensity of cell populations (planktonic and biofilm) in rabbit ileal loop fluids to form CVEC-L, we tested isolated cell populations for their ability to survive in water and form CVEC-L (Fig. 2B). Generally, the clumped or biofilm cells survived in water in a readily culturable form for a significantly longer duration than the planktonic cells (Fig. 2B). Once cells were no longer viable by conventional culture, these biofilm-associated cells remained recoverable as CVEC-L for a minimum of 60 days (Fig. 2A). In contrast, when planktonic cells were added to water, they did not form CVEC-L and the cell counts dropped rapidly with no live cells left within a few days (<10 days). These results suggested that biofilm development is a prerequisite to CVEC-L formation.

Fig. 2.

Development of the CVEC form by strain C6706 and its derivatives. (A) Conversion of V. cholerae in ileal loop fluids of rabbits to CVEC on inoculation into microcosms composed of filter-sterilized environmental water. (B) Mean duration of persistence of culturable cells after inoculation of planktonic cells or biofilms formed in vitro and in the ileal loops of rabbits into filter-sterilized environmental water at room temperature. The bars marked with an asterisk correspond to cell populations that were able to form CVEC, as determined by the AST procedure (Table S1). The duration of the survival of the clumped cells was longer compared to the planktonic cells for C6706WT and its hapR and hapA mutants. The mean and standard deviation of five independent observations are shown. Statistical test results compare the duration of the survival of planktonic and clumped cells. The two-tailed p values from Mann–Whitney tests are shown below the bar graph.

V. cholerae Biofilms Shed by Cholera Victims Convert to CVEC-L on Introduction into Water.

Because V. cholerae recovered from the intestine of experimentally challenged rabbits were found to convert to CVEC-L when inoculated in water, we decided to further verify this phenomenon with wild-type V. cholerae strains excreted by cholera victims. V. cholerae in the stools of seven different cholera patients were tested for conversion to CVEC. Planktonic and clumped cell fractions of the cholera stools were inoculated in filter-sterilized environmental water samples and monitored for CVEC formation by the AST procedure (17). These naturally occurring strains causing the recent epidemic of cholera are known to be resistant to streptomycin and suphamethoxazole–trimethoprim (SXT) due to the presence of the SXT element (21) and to nalidixic acid due to a chromosomal mutation. Thus, by definition, the CVEC form would not yield V. cholerae colonies in the AST assay (17) if pre-enriched in the presence of streptomycin but would produce colonies when pre-enriched in the presence of nalidixic acid, although the isolated bacteria exhibit resistance to both these antibiotics. In this assay, the presence of V. cholerae cells could be detected by the AST culture method up to 45 days postinoculation with clumped cells. However, planktonic cells could not be recovered after 9 days (Fig. 3). Thus the rabbit-derived V. cholerae cells and V. cholerae excreted by patients with natural infection exhibited similar behavior when inoculated in samples of environmental water.

Naturally Occurring CVEC Are Derived from Stools of Cholera Victims.

Because V. cholerae cells shed in stools of cholera patients or after passage through the rabbit intestine readily formed CVEC-L when inoculated in water, and because previous studies have indicated that the human host contributes to the epidemic spread of cholera by producing a hyper-infectious form of the pathogen (6, 10), we further analyzed naturally occurring CVEC of V. cholerae O1 in lake and river water to understand their origin. Limit dilution using a fluctuation analysis format allowed us to obtain multiple colonies derived from single clumps of naturally occurring CVEC. In brief, water samples were initially diluted 50-fold in bile–peptone medium, and the diluted samples were broken down further into different small aliquots. Each aliquot was assayed for the presence of CVEC and the appearance of viable cells on enrichment using the AST procedure (17). The extensive fractionation of diluted samples into many aliquots in the fluctuation test allowed a redistribution of possible CVEC clumps in water so that an aliquot of water would contain a maximum of one clump, whereas most aliquots would not contain any. Of 163 fractions of diluted water representing 15 surface-water samples analyzed, only 6 aliquots were found to be positive for V. cholerae O1. These six aliquots came from a total of five different water samples. The other bacterial flora in the water were also extensively diluted in the fluctuation assay so that the fractions that contained the CVEC clump yielded almost exclusively V. cholerae and very few colonies of other bacteria after the enrichment in bile–peptone medium.

A total of 126 randomly picked colonies selected on plates without antibiotics were subjected to comparative analysis. All 126 isolates were found to belong to a single ribotype of the O1 serogroup of V. cholerae and were resistant to SXT, streptomycin, and nalidixic acid (Fig. S3). The ribotype and antibiogram of these isolates also agreed with those of the recent predominant clone of V. cholerae associated with cholera epidemics in Bangladesh. We reasoned that, if the cellular aggregates were formed from bacteria growing in the environment, multiple clones of environmental V. cholerae or even more than one species of bacteria would be found in the cell clumps. The isolation of V. cholerae with identical properties in these limit dilution assays suggests that each clump of naturally occurring CVEC analyzed in this study were composed of a single clone of V. cholerae, which was also the predominant pathogenic clone causing cholera locally at that same time. These results strongly suggest that the clumps of CVEC present in the water were most likely derived from V. cholerae excreted by cholera victims.

Discussion

The role of CVEC in the epidemiology of cholera appears to be crucial because these cells can revert to fully active bacteria and presumably cause outbreaks of the disease (6). Our data show that strains capable of producing biofilms can form CVEC-L, whereas mutants that are impaired in the development of biofilms are unable to form CVEC-L when inoculated in water (Fig. 2 and Table S1). These observations clearly suggest that, if bona fide CVEC are in fact formed by mechanisms analogous to that of CVEC-L formation, then the development of biofilm before introduction into environmental water is an essential step in formation of natural CVEC. The most likely place for this to occur is within infected cholera victims.

In addition to demonstrating that formation of biofilms is a prerequisite to the development of CVEC, our studies have also provided parameters to clearly distinguish between these two forms. By definition, CVEC develop when V . cholerae become metabolically less active and unable to form colonies on nutrient agar plates, unless the cells are preincubated in nutrient broth for at least 5 h. In this form, the bacteria also become unable to express resistance to certain aminoglycoside antibiotics (for which the bacteria must produce aminoglycosidase enzymes to inactivate the antibiotics) despite carrying the resistance genes. Consequently, the bacteria cannot survive in the presence of these antibiotics unless they are preincubated in antibiotic-free nutrient medium. In the present study, the phenotypic indicator that was conveniently used to define this state was based on the ability of the bacteria (carrying the resistance genes) to express resistance to kanamycin or streptomycin, unless the resistance was due to chromosomal mutations. Whereas biofilm cells retained the ability to express resistance to these antibiotics, the CVEC form transiently lost the ability to inactivate aminoglycoside antibiotics, despite carrying the resistance genes, and became sensitive. We predict that the proposed VBNC cells(16) should also behave similarly, but there has not been any reported observation on the drug-resistance behavior of VBNC cells, simply because these cells, as reported, could not be cultured.

Possible genetic parameters for defining CVEC are still inadequate. However, our efforts to produce CVEC under laboratory conditions, as described in this study, are beginning to establish parameters to more conclusively define the CVEC form. Eventually, microarray-based expression profiling should be possible using laboratory-made CVEC. Thus our efforts to model CVEC formation in the laboratory should facilitate further studies of the properties of this survival form.

In many bacterial pathogens, biofilm formation and/or virulence factor production are induced at high cell density in the presence of quorum-sensing autoinducers (22, 23). In contrast, expression of virulence and biofilm formation by V. cholerae is repressed at high cell density (1 ⇓ ⇓–4). Recently, it has been suggested that quorum sensing plays a role in the dissolution of the “in vivo-formed biofilms” and thus constitutes an exit strategy of the bacteria toward the end of an infection cycle (3, 4). The clumps of cells found in cholera stools or in the intestinal loops of rabbits likely represent aggregates of cells derived from in vivo-formed biofilms. However, suspension of such clumps in a large volume of environmental water would be predicted to dilute extracellular autoinducers and thus provide a signal to remain associated as a biofilm rather than to initiate a process of becoming planktonic cells. This strategy is attractive because conversion of these clumps to planktonic forms would be detrimental to the bacteria if in fact CVEC formation is essential for aquatic survival and human transmission of the most successful clones of V. cholerae. It is therefore not surprising that many naturally occurring isolates of V. cholerae carry null mutations in hapR (2), which, on the basis of data presented here, would be predicted to both enhance virulence gene expression and EPS-dependent biofilm formation in vivo and promote robust CVEC formation after being shed from the host into environmental waters. A single clump of V. cholerae CVEC potentially represents an infectious human oral dose, given their high cell count and the ability of CVEC to be resuscitated in vivo (3, 6).

In conclusion, we have shown that CVEC, a conditionally viable form of V. cholerae present in environmental water samples in cholera-endemic areas, correspond to clonal communities of cells that are identical to those derived from stools of cholera victims. We have developed a laboratory procedure for the production of cells that mimic natural V. cholerae CVEC and have shown that this quiescent form of V. cholerae is most readily formed when animal-derived V. cholerae organized in biofilm-like communities are challenged with survival in environmental water. Mutations that enhance or block the formation of biofilms similarly enhance or block the formation of CVEC under laboratory conditions. Studies by Merrell et al. (10) provided evidence that passage through the human host alters critical phenotypic properties that enhance the infectivity of V. cholerae in experimental animals. One of these is the down-regulation of motility/chemotaxis, which interestingly has been linked to the up-regulation of EPS production and thus biofilm formation (2, 11, 24). Our studies indicate that the host-induced changes in V. cholerae also include induction of in vivo biofilm communities that are especially efficient at CVEC formation and are therefore capable of long-term survival when introduced into aquatic environments. Shedding of contaminated stools by cholera victims into environmental water thus provides multiple mechanisms for enhancing the efficient transmission of cholera under endemic and epidemic conditions.

Materials and Methods

Bacterial Strains and Specimens.

Relevant characteristics of V. cholerae strains used in this study to investigate the development of biofilms and CVEC are listed in Table S2. The transposon insertion mutants were isolated from a TnFGL3 insertion library of strain C6706 described previously (20).

The International Centre for Diarrheal Disease Research in Bangladesh (ICDDRB) maintains a cholera surveillance system at its Dhaka hospital; cholera stool samples used in the study were obtained from these patients. Specimens from these patients were also used to obtain clinical V. cholerae as required for the studies described. Protocols for studies involving human specimens were reviewed and approved by the Research Review Committee and Ethical Review Committee at the ICDDRB and by the institutional review boards at Harvard Medical School and Massachusetts General Hospital.

The environmental V. cholerae strains were isolated from surface waters in Dhaka. For estimation of naturally occurring CVEC and construction of microcosms, water samples were collected from one or more previously described sampling sites in and around Dhaka city (7).

Detection of V. cholerae CVEC.

The presence of CVEC of V. cholerae O1 was estimated by exploiting the antibiotic resistance property of the bacteria, using the AST procedure described previously (17). The AST procedure was based on the observation that V. cholerae strains causing recent cholera epidemics in Bangladesh carry the SXT element (21), which encodes resistance to multiple antibiotics, including streptomycin (Sm^R), sulfamethoxazole, and trimethoprim (SXT^R). In these strains, Sm^R is mediated through enzymatic modification of the antibiotic by enzymes encoded by the strAB genes of the SXT element (21, 25). In addition, the strains are also resistant to nalidixic acid arising from mutations in the gyrA gene encoding the GyrA subunit of DNA gyrase (26). By definition, CVEC of V. cholerae were unable to express Sm^R that requires protein synthesis unless they were revitalized by initially culturing in the absence of streptomycin. However, the CVEC form remained resistant to nalidixic acid.

Briefly, an aliquot (5.0 mL) of each water sample was added to 2.5 mL of 3× concentrated bile–peptone medium (BP; 1% peptone, 0.5% taurocholic acid, 1% NaCl, pH 9.0) and incubated for 5 h for enrichment of V. cholerae. In another set of BP enrichment cultures, an antibiotic, i.e., streptomycin (70 μg/mL) or nalidixic acid (30 μg/mL), was added. Dilutions of the enrichment cultures were spread on taurocholate tellurite gelatin agar (TTGA) plates (27) containing streptomycin (70 μg/mL) and on TTGA plates devoid of any antibiotic. Suspected Vibrio colonies were picked and subjected to standard biochemical and serological tests (28). By definition, samples containing naturally occurring CVEC of recent epidemic strains would not yield V. cholerae colonies on TTGA plates if pre-enriched in the presence of streptomycin but would produce V. cholerae colonies when pre-enriched in the presence of nalidixic acid, although isolated strains exhibit resistance to both these antibiotics.

In contrast, the mutant strains analyzed in this study carried a kanamycin resistance (Kn^R) cassette encoding aminoglycoside phosphotransferase [due to the aphA-2 gene present on transposon TnFGL3 used in construction of the library (20)] and a constitutive Sm^R (due to chromosomal mutations in the ribosomal protein S12). Therefore, in the CVEC-L state, the mutants would become sensitive to kanamycin despite carrying the aphA gene; i.e., Kn^R would be lost but Sm^R would be retained by the CVEC-L form of the mutants.

Fluctuation Test and Strain Composition of Natural CVEC.

The fluctuation assay was conducted to obtain multiple colonies derived from possible single clumps of naturally occurring CVEC and then to analyze these isolates to determine whether the clumps comprised single or multiple clones of V. cholerae. In the fluctuation assay, water samples were initially diluted 50-fold in BP medium, and the diluted samples were broken down further into different small aliquots. Each aliquot was assayed for the appearance of viable cells by a growth kinetics assay. Briefly, 100-μl aliquots of samples were removed from the enrichment mixture of a typical AST procedure (17) at regular intervals and plated on antibiotic-containing selective medium for the target V. cholerae strain as well as on medium without antibiotic. Suspected Vibrio colonies (gelatinase positive) were randomly picked from TTGA plates without antibiotic (10–30 colonies from each positive enrichment culture in the fluctuation test) together with two to three colonies from the antibiotic-containing plates. These isolates were subjected to analysis of serotype, antimicrobial susceptibility, virulence gene content, and ribotype (29) to determine clonal relationships.

Antimicrobial Resistance:

All V. cholerae isolates were tested for antimicrobial resistance by the method of Bauer et al. (30) using standard antibiotic disks (Oxoid) at the following antibiotic concentrations (μg/disk): ampicillin, 10; chloramphenicol, 30; streptomycin, 10; tetracycline, 30; trimethoprim–sulfamethoxazole, 1.25 and 23.75, respectively; kanamycin, 30; gentamicin, 10; ciprofloxacin, 5; norfloxacin, 10; and nalidixic acid, 30.

Probes and PCR Assays.

The presence of virulence genes was determined by using specific PCR assays for the tcpA, tcpI, and acfB genes of the toxin coregulated pilus pathogenicity island (31) and the ctxA and zot genes of the CTX prophage (31, 32). The SXT probe and the rRNA gene probe used in ribotyping have also been described previously (21, 29). The toxR probe used to quantify clumped V. choleare cells in rabbit fluids was a 2.4-kb BamHI fragment of pVM7 (33) Southern blots, and dot blots were prepared and hybridized with radioactively labeled probes following standard methods (34).

Estimation of Biofilm Development.

Quantification of biofilm formation by different strains was done by spectrophotometry of crystal-violet-stained biofilm cells as described previously (2, 3). Briefly, overnight cultures of V. cholerae were diluted into 1 mL of LB medium in autoclaved 12- × 75-mm borosilicate glass tubes and incubated at room temperature without agitation. After the indicated time, planktonic cells were washed away with distilled water and the remaining biofilm-associated cells were stained with 1% crystal violet. To quantify biofilm formation, the tubes were rinsed three times, and stained biofilms were solubilized with 1 mL of dimethyl sulfoxide, and an OD at 570 nm was determined.

In addition, observation of live biofilms formed both in vitro and in the ileal loops of rabbits was conducted by phase-contrast and fluorescent microscopy. The cells were rendered fluorescent due to the presence of the GFPmut3 gene in the transposon TnFGL3 used in construction of the mutants (20) or because of a plasmid expressing GFP introduced into the wild-type strain C6706.

Assays in Cholera Stools.

Freshly collected cholera stools from patients who did not receive antibiotics before reporting to the hospital were examined by dark-field microscopy to ascertain the presence of typical highly motile vibrio-shaped organisms. The stools were diluted in environmental water that had previously tested negative for V. cholerae O1 or O139 by the AST procedure (17). An aliquot of this dilution and a dilution of stools in PBS were cultured immediately to calculate the initial number of V. cholerae organisms present in the stools. Because stools from cholera patients are known to contain lytic vibriophages, the presence of phage was also tested by previously described methods (7). In this study, we used stools that were found negative for any lytic phage. The stool-spiked water was left at room temperature, and aliquots were periodically removed and cultured to obtain cell counts. When the water became apparently negative for V. cholerae by standard culture, the samples were tested by the AST procedure as described for environmental water samples to determine the presence of CVEC.

Similar assays were also conducted using planktonic and clumped cell populations isolated from human cholera stools. Stools were fractionated according to the size of suspended particles by centrifugation, initially at 2000 × g to precipitate clumped cells and debris, whereas the supernatant contained mostly planktonic cells. The precipitate was further washed in PBS (pH 7.2) to remove any loosely adhering cells and finally resuspended in fresh PBS. Relative survival and CVEC formation by these two populations of cells were studied using methods described above.

Assays in Rabbits.

A set of transposon insertion mutants of strain C6706 and the parent strain were tested for their ability to develop CVEC (Table S1) when inoculated into microcosms composed of filter-sterilized environmental water after passage in the ileal loops of adult rabbits (19). New Zealand White rabbits obtained from the breeding facilities of the ICDDRB were used. A maximum of six ileal loops of ≈10 cm in length were made in each rabbit, which had previously been fasted for 48 h. Overnight cultures of each V. cholerae strain were diluted 1:100-fold in fresh LB medium and grown for 2 h at 37°C with shaking. Cells were precipitated by centrifugation and resuspended in 10 mM PBS, pH 7.4, at a concentration of ≈1 × 10⁹ cells/ml. One milliliter of the bacterial suspension was inoculated into each loop as described previously (19). The same strain was tested in at least five different rabbits. After 18 h, rabbits were killed and the ileal loop fluid was collected. The fluid was examined under fluorescent and phase-contrast microscopy. Dilutions of the ileal loop fluid were inoculated into microcosms composed of filter-sterilized environmental water and left at room temperature. Samples were periodically removed and cultured for the presence of V. cholerae. When the water became culture negative, the samples were further tested by the AST procedure (17) to ascertain the presence of CVEC of the relevant V. cholerae strain. Similar assays were also conducted using planktonic and clumped cells isolated from the rabbit ileal loop fluids, as described for assays in cholera stools.

Estimation of V. cholerae in Rabbit Ileal Loop Fluids.

The clumped cells were separated and washed by differential centrifugation as described previously (7), and the cells were resuspended in equal volumes of PBS. The suspension was diluted 10⁴-fold in fresh PBS. The diluted samples were further broken down into small aliquots (1.5 mL) and were subjected to total DNA extraction using standard methods. Dot blots were prepared by diluting the DNA samples from each aliquot together with dilutions of DNA extracted from a reconstituted sample with a known number of V. cholerae cells spiked into PBS. The dot blots were hybridized with a radioactively labeled toxR probe, and autoradiographs were developed. The intensity of the hybridization of each dot was compared with the known standard to estimate the number of V. cholerae cells. In addition, cells were also estimated by standard dilution culture in which dilutions of fractions produced as above were cultured after vortexing in the presence of glass beads to break the cell clumps.

Statistical Analysis.

Statistical comparison between two sets of data were carried out by the Mann–Whitney test. Differences were considered to be significant when the two-tailed p was ≤0.05. Data analyses were done by using a statistical software (Winstat for MS Excel, version 2009.1).

Acknowledgments

This research was funded in part by National Institutes of Health grants 2R01-GM068851-5, AI070963-01A1, and U01-AI058935 under different sub-agreements between the Harvard Medical School, Massachusetts General Hospital, and the International Centre for Diarrheal Disease Research in Bangladesh. The International Centre for Diarrheal Disease Research in Bangladesh is supported by countries and agencies that share its concern for the health problems of developing countries.

Footnotes

↵ ¹To whom correspondence may be addressed at: International Centre for Diarrhoeal Disease Research, Mohakhali, Dhaka 1212, Bangladesh. E-mail: faruque{at}icddrb.org, john_mekalanos{at}hms.harvard.edu, gbnair_2000{at}yahoo.com.

Author contributions: G.B.N., J.J.M., and S.M.F. designed research; M.K., S.M.N.U., D.E.C., and S.M.F. performed research; S.B.C., J.J.M., and S.M.F. analyzed data; and S.B.C., J.J.M., and S.M.F. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0913404107/DCSupplemental.

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1. Butler SM,
2. et al.
(2006) Cholera stool bacteria repress chemotaxis to increase infectivity. Mol Microbiol 60:417–426.
OpenUrl CrossRef PubMed
↵
1. Snow J
(1855) On the Mode of Communication of Cholera (J. Churchill, London), , 2nd Ed.
↵
1. Kaper JB,
2. Morris JG Jr,
3. Levine MM
(1995) Cholera. Clin Microbiol Rev 8:48–86.
OpenUrl Abstract/FREE Full Text
↵
1. Khan MU,
2. Shahidullah MD,
3. Haque MS,
4. Ahmed WU
(1984) Presence of vibrios in surface water and their relation with cholera in a community. Trop Geogr Med 36:335–340.
OpenUrl PubMed
↵
1. Cash RA,
2. et al.
(1974) Response of man to infection with Vibrio cholerae. I. Clinical, serologic, and bacteriologic responses to a known inoculum. J Infect Dis 129:45–52.
OpenUrl Abstract/FREE Full Text
↵
1. Wachsmuth IK,
2. Blake PA,
3. Olsvik O
1. Colwell RR,
2. Huq A
(1994) in Vibrio cholerae and Cholera: Molecular to Global Perspectives, eds Wachsmuth IK, Blake PA, Olsvik O(ASM Press, Washington, DC), pp 117–133.
↵
1. Faruque SM,
2. et al.
(2006) An improved technique for isolation of environmental Vibrio cholerae with epidemic potential: Monitoring the emergence of a multiple-antibiotic-resistant epidemic strain in Bangladesh. J Infect Dis 193:1029–1036.
OpenUrl Abstract/FREE Full Text
↵
1. Nelson ET,
2. Clements JD,
3. Finkelstein RA
(1976) Vibrio cholerae adherence and colonization in experimental cholera: Electron microscopic studies. Infect Immun 14:527–547.
OpenUrl Abstract/FREE Full Text
↵
1. De SN,
2. Chatterje DN
(1953) An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J Pathol Bacteriol 66:559–562.
OpenUrl CrossRef PubMed
↵
1. Cameron DE,
2. Urbach JM,
3. Mekalanos JJ
(2008) A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae Proc Natl Acad Sci USA 105:8736–8741.
OpenUrl Abstract/FREE Full Text
↵
1. Waldor MK,
2. Tschäpe H,
3. Mekalanos JJ
(1996) A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139. J Bacteriol 178:4157–4165.
OpenUrl Abstract/FREE Full Text
↵
1. Surette MG,
2. Bassler BL
(1998) Quorum sensing in Escherichia coli and Salmonella typhimurium Proc Natl Acad Sci USA 95:7046–7050.
OpenUrl Abstract/FREE Full Text
↵
1. Surette MG,
2. Miller MB,
3. Bassler BL
(1999) Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: A new family of genes responsible for autoinducer production. Proc Natl Acad Sci USA 96:1639–1644.
OpenUrl Abstract/FREE Full Text
↵
1. Martinez-Wilson HF,
2. Tamayo R,
3. Tischler AD,
4. Lazinski DW,
5. Camilli A
(2008) The Vibrio cholerae hybrid sensor kinase VieS contributes to motility and biofilm regulation by altering the cyclic diguanylate level. J Bacteriol 190:6439–6447.
OpenUrl Abstract/FREE Full Text
↵
1. Hochhut B,
2. et al.
(2001) Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob Agents Chemother 45:2991–3000.
OpenUrl Abstract/FREE Full Text
↵
1. Walsh C
(2003) Antibiotics: Actions, Origins, Resistance (ASM Press, Washington, DC), pp 89–156.
↵
1. Monsur KA
(1961) A highly selective gelatin-taurocholate-tellurite medium for the isolation of Vibrio cholerae. Trans R Soc Trop Med Hyg 55:440–442.
OpenUrl CrossRef PubMed
↵
1. World Health Organization
(1974) World Health Organization guidelines for the laboratory diagnosis of cholera (Bacterial Disease Unit, WHO, Geneva).
↵
1. Faruque SM,
2. Roy SK,
3. Alim ARMA,
4. Siddique AK,
5. Albert MJ
(1995) Molecular epidemiology of toxigenic Vibrio cholerae in Bangladesh studied by numerical analysis of rRNA gene restriction patterns. J Clin Microbiol 33:2833–2838.
OpenUrl Abstract/FREE Full Text
↵
1. Bauer AW,
2. Kirby WMM,
3. Sherris JC,
4. Turck M
(1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496.
OpenUrl PubMed
↵
1. Faruque SM,
2. et al.
(04) Genetic diversity and virulence potential of environmental Vibrio cholerae population in a cholera-endemic area. Proc Natl Acad Sci USA 101:2123–2128.
OpenUrl CrossRef
↵
1. Waldor MK,
2. Mekalanos JJ
(1996) Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910–1914.
OpenUrl Abstract/FREE Full Text
↵
1. Miller VL,
2. Mekalanos JJ
(1984) Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc Natl Acad Sci USA 81:3471–3475.
OpenUrl Abstract/FREE Full Text
↵
1. Maniatis T,
2. Fritsch EF,
3. Sambrook J
(1982) Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

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Proceedings of the National Academy of Sciences: 107 (4)

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Article Classifications

Biological Sciences
Microbiology

[1] ↵
Cámara M,
Hardman A,
Williams P,
Milton D
(2002) Quorum sensing in Vibrio cholerae Nat Genet 32:217–218.
OpenUrl CrossRef PubMed

[2] Cámara M,

[3] Hardman A,

[4] Williams P,

[5] Milton D

[6] ↵
Hammer BK,
Bassler BL
(2003) Quorum sensing controls biofilm formation in Vibrio cholerae Mol Microbiol 50:101–104.
OpenUrl CrossRef PubMed

[7] Hammer BK,

[8] Bassler BL

[9] ↵
Zhu J,
Mekalanos JJ
(2003) Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae Dev Cell 5:647–656.
OpenUrl CrossRef PubMed

[10] Zhu J,

[11] Mekalanos JJ

[12] ↵
Zhu J,
et al.
(2002) Quorum-sensing regulators control virulence gene expression in Vibrio cholerae Proc Natl Acad Sci USA 99:3129–3134.
OpenUrl Abstract/FREE Full Text

[13] Zhu J,

[14] et al.

[15] ↵
Faruque SM,
Albert MJ,
Mekalanos JJ
(1998) Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae Microbiol Mol Biol Rev 62:1301–1314.
OpenUrl Abstract/FREE Full Text

[16] Faruque SM,

[17] Albert MJ,

[18] Mekalanos JJ

[19] ↵
Faruque SM,
et al.
(2006) Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proc Natl Acad Sci USA 103:6350–6355.
OpenUrl Abstract/FREE Full Text

[20] Faruque SM,

[21] et al.

[22] ↵
Faruque SM,
et al.
(2005) Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage. Proc Natl Acad Sci USA 102:6119–6124.
OpenUrl Abstract/FREE Full Text

[23] Faruque SM,

[24] et al.

[25] ↵
Faruque SM,
et al.
(2005) Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages. Proc Natl Acad Sci USA 102:1702–1707.
OpenUrl Abstract/FREE Full Text

[26] Faruque SM,

[27] et al.

[28] ↵
Nelson EJ,
Harris JB,
Morris JG Jr,
Calderwood SB,
Camilli A
(2009) Cholera transmission: The host, pathogen and bacteriophage dynamic. Nat Rev Microbiol 7:693–702.
OpenUrl CrossRef PubMed

[29] Nelson EJ,

[30] Harris JB,

[31] Morris JG Jr,

[32] Calderwood SB,

[33] Camilli A

[34] ↵
Merrell DS,
et al.
(2002) Host-induced epidemic spread of the cholera bacterium. Nature 417:642–645.
OpenUrl CrossRef PubMed

[35] Merrell DS,

[36] et al.

[37] ↵
Butler SM,
et al.
(2006) Cholera stool bacteria repress chemotaxis to increase infectivity. Mol Microbiol 60:417–426.
OpenUrl CrossRef PubMed

[38] Butler SM,

[39] et al.

[40] ↵
Snow J
(1855) On the Mode of Communication of Cholera (J. Churchill, London), , 2nd Ed.

[41] Snow J

[42] ↵
Kaper JB,
Morris JG Jr,
Levine MM
(1995) Cholera. Clin Microbiol Rev 8:48–86.
OpenUrl Abstract/FREE Full Text

[43] Kaper JB,

[44] Morris JG Jr,

[45] Levine MM

[46] ↵
Khan MU,
Shahidullah MD,
Haque MS,
Ahmed WU
(1984) Presence of vibrios in surface water and their relation with cholera in a community. Trop Geogr Med 36:335–340.
OpenUrl PubMed

[47] Khan MU,

[48] Shahidullah MD,

[49] Haque MS,

[50] Ahmed WU

[51] ↵
Cash RA,
et al.
(1974) Response of man to infection with Vibrio cholerae. I. Clinical, serologic, and bacteriologic responses to a known inoculum. J Infect Dis 129:45–52.
OpenUrl Abstract/FREE Full Text

[52] Cash RA,

[53] et al.

[54] ↵
Wachsmuth IK,
Blake PA,
Olsvik O
Colwell RR,
Huq A
(1994) in Vibrio cholerae and Cholera: Molecular to Global Perspectives, eds Wachsmuth IK, Blake PA, Olsvik O(ASM Press, Washington, DC), pp 117–133.

[55] Wachsmuth IK,

[56] Blake PA,

[57] Olsvik O

[58] Colwell RR,

[59] Huq A

[60] ↵
Faruque SM,
et al.
(2006) An improved technique for isolation of environmental Vibrio cholerae with epidemic potential: Monitoring the emergence of a multiple-antibiotic-resistant epidemic strain in Bangladesh. J Infect Dis 193:1029–1036.
OpenUrl Abstract/FREE Full Text

[61] Faruque SM,

[62] et al.

[63] ↵
Nelson ET,
Clements JD,
Finkelstein RA
(1976) Vibrio cholerae adherence and colonization in experimental cholera: Electron microscopic studies. Infect Immun 14:527–547.
OpenUrl Abstract/FREE Full Text

[64] Nelson ET,

[65] Clements JD,

[66] Finkelstein RA

[67] ↵
De SN,
Chatterje DN
(1953) An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J Pathol Bacteriol 66:559–562.
OpenUrl CrossRef PubMed

[68] De SN,

[69] Chatterje DN

[70] ↵
Cameron DE,
Urbach JM,
Mekalanos JJ
(2008) A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae Proc Natl Acad Sci USA 105:8736–8741.
OpenUrl Abstract/FREE Full Text

[71] Cameron DE,

[72] Urbach JM,

[73] Mekalanos JJ

[74] ↵
Waldor MK,
Tschäpe H,
Mekalanos JJ
(1996) A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139. J Bacteriol 178:4157–4165.
OpenUrl Abstract/FREE Full Text

[75] Waldor MK,

[76] Tschäpe H,

[77] Mekalanos JJ

[78] ↵
Surette MG,
Bassler BL
(1998) Quorum sensing in Escherichia coli and Salmonella typhimurium Proc Natl Acad Sci USA 95:7046–7050.
OpenUrl Abstract/FREE Full Text

[79] Surette MG,

[80] Bassler BL

[81] ↵
Surette MG,
Miller MB,
Bassler BL
(1999) Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: A new family of genes responsible for autoinducer production. Proc Natl Acad Sci USA 96:1639–1644.
OpenUrl Abstract/FREE Full Text

[82] Surette MG,

[83] Miller MB,

[84] Bassler BL

[85] ↵
Martinez-Wilson HF,
Tamayo R,
Tischler AD,
Lazinski DW,
Camilli A
(2008) The Vibrio cholerae hybrid sensor kinase VieS contributes to motility and biofilm regulation by altering the cyclic diguanylate level. J Bacteriol 190:6439–6447.
OpenUrl Abstract/FREE Full Text

[86] Martinez-Wilson HF,

[87] Tamayo R,

[88] Tischler AD,

[89] Lazinski DW,

[90] Camilli A

[91] ↵
Hochhut B,
et al.
(2001) Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob Agents Chemother 45:2991–3000.
OpenUrl Abstract/FREE Full Text

[92] Hochhut B,

[93] et al.

[94] ↵
Walsh C
(2003) Antibiotics: Actions, Origins, Resistance (ASM Press, Washington, DC), pp 89–156.

[95] Walsh C

[96] ↵
Monsur KA
(1961) A highly selective gelatin-taurocholate-tellurite medium for the isolation of Vibrio cholerae. Trans R Soc Trop Med Hyg 55:440–442.
OpenUrl CrossRef PubMed

[97] Monsur KA

[98] ↵
World Health Organization
(1974) World Health Organization guidelines for the laboratory diagnosis of cholera (Bacterial Disease Unit, WHO, Geneva).

[99] World Health Organization

[100] ↵
Faruque SM,
Roy SK,
Alim ARMA,
Siddique AK,
Albert MJ
(1995) Molecular epidemiology of toxigenic Vibrio cholerae in Bangladesh studied by numerical analysis of rRNA gene restriction patterns. J Clin Microbiol 33:2833–2838.
OpenUrl Abstract/FREE Full Text

[101] Faruque SM,

[102] Roy SK,

[103] Alim ARMA,

[104] Siddique AK,

[105] Albert MJ

[106] ↵
Bauer AW,
Kirby WMM,
Sherris JC,
Turck M
(1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496.
OpenUrl PubMed

[107] Bauer AW,

[108] Kirby WMM,

[109] Sherris JC,

[110] Turck M

[111] ↵
Faruque SM,
et al.
(04) Genetic diversity and virulence potential of environmental Vibrio cholerae population in a cholera-endemic area. Proc Natl Acad Sci USA 101:2123–2128.
OpenUrl CrossRef

[112] Faruque SM,

[113] et al.

[114] ↵
Waldor MK,
Mekalanos JJ
(1996) Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910–1914.
OpenUrl Abstract/FREE Full Text

[115] Waldor MK,

[116] Mekalanos JJ

[117] ↵
Miller VL,
Mekalanos JJ
(1984) Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc Natl Acad Sci USA 81:3471–3475.
OpenUrl Abstract/FREE Full Text

[118] Miller VL,

[119] Mekalanos JJ

[120] ↵
Maniatis T,
Fritsch EF,
Sambrook J
(1982) Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[121] Maniatis T,

[122] Fritsch EF,

[123] Sambrook J

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