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1364 Vibrio cholerae 01 Can Assume a Chlorine-Resistant Rugose Survival Form that Is Virulent for Humans J. Glenn Morris, Jr., Marcelo B. Sztein, Eugene W. Rice, James P. Nataro, Genevieve A. Losonsky, Pinaki Panigrahi, Carol O. Tacket, and Judith A. Johnson Center for Vaccine Development, Divisions of Infectious Diseases (Department of Medicine) and of Neonatology (Department of Pediatrics), and Department of Pathology, University of Maryland School of Medicine, and Veterans Affairs Medical Center, Baltimore, Maryland; Drinking Water Research Division, Risk Reduction Engineering Laboratory, US Environmental Protection Agency, Cincinnati. Ohio Vibrio cholerae can shift to a "rugose" colonial morphology associated with expression of an amorphous exopolysaccharide that promotes cell aggregation. Flow cytometric studies indicated that up to 3% of particles in rugose cultures represented aggregates of >5 bacterial cells. Rugose variants of our test strains displayed resistance to killing by chlorine, with viable cells persisting for >30 min in 2 mg/L free chlorine; strains also showed resistance to killing by complement-mediated serum bactericidal activity. Six volunteers fed 106 cfu of a rugose variant of V. cholerae 01 EI Tor Inaba N16961 developed symptoms typical of cholera, with a mean diarrheal stool volume of 2.2 L (range, 1.4-4.3). Isolates recovered from the stool of infected volunteers retained the rugose phenotype. The data suggest that rugose strains cause human disease. The role of these strains in the epidemiology of cholera remains to be determined. Researchers working with Vibrio cholerae have traditionally selected smooth colonies from culture plates for study; little attention has been given to the pathogenesis and survival of other colonial morphologies, most of which have been dismissed as avirulent rough variants. In 1938, Bruce White at the National Institute for Medical Research (London) provided a detailed description of "rugose," or wrinkled, colonies that appeared on serial passage of smooth V. cholerae strains [1] (figure 1). In a series of studies conducted in the 1930s and 1940s, he demonstrated that cells from rugose colonies were embedded in an amorphous intercellular matrix (which he termed zoogloea) and were distinct morphologically and immunologically from "rough" variants, which have a modified lipopolysaccharide (LPS) [1-3]. Interest in rugose forms of V. cholerae was revived during the recent South American epidemics with the demonstration by E. W. Rice and colleagues at the US Environmental Protection Agency that bacteria within rugose cultures remained via- Received 5 March 1996; revised 16 July 1996. Presented in part: 29th Joint Conference on Cholera and Related Diarrheal Diseases, Asilomar, Calfomia, 1-3 December 1993. All studies were approved by the Institutional Review Board, University of Maryland at Baltimore. Informed consent was obtained from all study participants. Financial support: Thrasher Research Fund (to J.G.M.) and Department of Veterans Affairs (to J.A.J.) for laboratory studies; NIH (AI-15096) for volunteer studies. Reprints or correspondence: Dr. J. Glenn Morris, Jr., Infectious Diseases Section, Veterans Affairs Medical Center, 10 N. Greene St., Baltimore, MD 21201. The Journal of Infectious Diseases 1996; 174:1364-8 © 1996 by The University of Chicago. All rights reserved. 0022-1899/96/7406-0035$01.00 ble in the presence of chlorine. Both smooth and rugose forms were found to be adherent to Caco-2 cells (a model for intestinal adherence), and both generated a striking fluid accumulation response in ligated rabbit ileal loop models, suggesting that smooth and rugose variants had comparable virulence [4]. However, the rugose phenotype remained poorly characterized, and there were uncertainties about the ability of rugose strains to actually colonize and cause illness in humans. Materials and Methods Characterization of rugose variants. Initial studies were done with 48 V cholerae strains, including 25 in a group 1 (V cholerae 01),9 in 0 group 139 (V cholerae 0139 Bengal), and 14 in other a groups (non-Ol V cholerae). Three strains were studied in more detail: V cholerae N16961, an EI Tor Inaba strain well characterized in animal models and human volunteers [5]; V cholerae C6706, an EI Tor Inaba strain from a cholera patient in Peru [4]; and V cholerae NRT-36S, an encapsulated 031 strain that we have shown to be pathogenic in animals and volunteers [6]. To isolate spontaneous rugose variants, bacteria from smooth colonies were passed in alkaline peptone water for 2-4 days and then plated on Luria agar at 37°C. Hiss staining was used to examine cells by light microscopy. For electron microscopy, cells were grown on CF A agar at 25°C (conditions that have previously been shown to optimize expression of pili in V. cholerae [7]) and stained with phosphotungstic acid. Cell aggregation was quantitated by flow cytometry (EPICS ELITE flow cytometer/cell sorter; Coulter Cytometry, Hialeah, FL). In these latter studies, data for 200,000-500,000 bacterial particles/sample were analyzed in a 128 X 128 matrix by using the Multi-2D software package (Phoenix Flow Systems, San Diego) and shown as posterior views of rotated forward-scatter versus side-scatter isometric displays. JID 1996; 174 (December) Concise Communications 1365 Figure 1. Photomicrograph of rugose colony of V. cholerae. Chlorine inactivation experiments (in triplicate) were done at 20 ± 2aC in pH 7.0 chlorine demand-free phosphate buffer at various concentrations of free chlorine [4, 8]. To evaluate resistance of rugose variants to complement-mediated killing by normal human serum, bacteria were incubated for 30 min with 65% pooled normal (nonimmune) human serum at 37°C in the presence of guinea pig complement [9]. Volunteer studies. Studies were done under quarantine on the inpatient Research Isolation Ward of the Center for Vaccine Development , University Hospital, following previously described protocols [6, 10]. Mean age of volunteers was 28 years (range, 19-39) and all were in excellent health as determined by comprehensive physical and laboratory examinations. The challenge strain was administered to the volunteers with 2 g of sodium bicarbonate to neutralize stomach acidity . All stools passed by the volunteers were characterized, weighed, and cultured for V. cholerae by direct plating after enrichment [6, 10]. Therapy with oral rehydration solution (1.5 mU1.0 g of liquid stool) was initiated immediately after passage ofthe first diarrheal stool. Therapy with tetracycline (to which both smooth and rugose variants were susceptible) was started 24 h after the onset of diarrhea. In preparing the bacterial inoculum, we picked 3 rugose colonies that were agglutinable with Inaba antiserum from brain-heart infusion (BHI) plates that had been inoculated with a pure frozen stock of a spontaneous rugose variant of V. cholerae strain N 16961. After overnight incubation at 37°C, the bacterial growth was harvested from all three BHI plates, washed twice in PBS, and resuspended in PBS, pH 7.4, with vigorous vortexing for I min. The concentration of the resultant suspension was adjusted spectrophotometrically, and counts were verified by direct plating. In samples collected on days 7 and 10, antibody-secreting cells were identified from among peripheral blood mononuclear cells by ELISPOT as previously described [10]; cells were screened for expression of IgG, IgM, and IgA directed against cholera toxin (CT), V. cholerae Inaba and Ogawa LPSs , and the rugose exopolysaccharide (isolated and partially purified by a modification of the method originally described by White [I D. In samples collected on days 7, 10, 21, and 28, specific serum IgG responses to CT and IgG, IgM, and IgA responses to Inaba and Ogawa LPSs, were measured by ELISA. Sera were also tested for vibriocidal activity against standard test strains (V. cholerae 01 El Tor Ogawa 3008 and V. cholerae 0 I classical Inaba VO I) and the challenge strain, V. cholerae N 16961/Ru . Methods and definitions for positive responses have been reported [6, 10, II]. Results Characterization of rugose variants. It was possible to identify rugose variants of all 48 V. cholerae strains studied. On light microscopy with a Hiss stain, rugose variants appeared as small coccoid shapes aggregated in a background of faintly stained material. In contrast, the smooth variants had normal vibrio morphology and little background material. When examined by electron microscopy, type C pili [7] were present on smooth and rugose variants of C6706; no pili were seen on Concise Communications 1366 JID 1996; 174 (December) N16961/Ru 1 1 SS Log FS Log N16961 Figure 2. Light-scatter properties of V cholerae strains N 16961 ( smooth) and N16961/Ru (rugose), as determined by flow cytometry. Data are shown as posterior views of rotated forward-scatter vs. side-scatter isometric displays . In experiment shown, % of aggregates was 0.4% for NI6961 and 2.8% for NI6961 /Ru . 1 SS Log FS Log either variant ofNRT-36S. Electron microscopic studies further demonstrated aggregation of bacterial cells in the rugose cultures, as well as a suggestion of an intercellular matrix material linking the cells. Flow cytometric techniques were used to quantitate cell aggregation. While aggregates were virtually nonexistent in smooth V. cholerae suspensions, up to 3% of the events collected from rugose bacterial suspensions represented bacterial aggregates (figure 2). To confirm that the major peak near the origin in the isometric display represented single bacteria, while the higher intensity forward-scatter and side-scatter particles represented bacterial aggregates, bacteria in the different regions were sorted (i.e., physically separated) and examined by Gram's stain. Small particles were within an electronic gate centered on the singles/ doubles peak shown in figure 2; aggregates were within a gate centered on the aggregate region. While the main peak near the origin in the isometric displays consisted primarily of single and double cells, most bacteria in the aggregates region consisted of 6 to >50 bacteria in an aggregate form; 0.9% of the aggregate particles counted had > 50 cells. Concise Communications JID 1996; 174 (December) 1367 bacterial count (log10) 7r----------------------------------. 6 5 4 3 2 1 O---. o -----.------l.-- 5 ..l-- 10 --L 15 ...i- 20 ...--J'---- 25 -.L. 30 ..........--J 35 minutes - - - N16961 smooth Figure 3. -+- N16961 rugose Survival of V cholerae El Tor N1696l (smooth) and N16961/Ru (rugose) in presence of 0.5 mg/L free chlorine. As shown in figure 3, cultures of smooth variants were consistently inactivated in <20 s when exposed to 0.5 mg/L free chlorine. In contrast, disinfection of rugose variants displayed a deviation from first-order kinetics, with an initial 2-3 order of magnitude decrease in the number of viable bacteria, followed by persistence of a subpopulation of cells. In an effort to totally eradicate rugose strains, bacteria were exposed to 2 mg/L free chlorine at pH 7.0 and 20°C; starting with an inoculum of ,....., 106 cfu, V. cholerae were still recoverable after 30 min. Smooth variants were completely killed when incubated for 30 min with normal (non immune) human serum in the presence of complement. Rugose strains survived in serum, although counts decreased from an initial inoculum of 107 . 1 to 104 . 1 for C6706 and from 106 .9 to 104 .3 for N16961. Volunteer studies. The spontaneous rugose variant of V. cholerae strain Nl6961 was administered to 6 volunteers at an inoculum of ~ 106 cfu/mL; when plated out, the inoculum was 99.83% rugose (1/600 colonies was smooth). All volunteers receiving the rugose strain developed profuse, watery diarrhea, with a mean incubation period of 19 h. Mean stool volume was 2.2 L, with volunteers having an average of 15.3 diarrheal stools. Peak excretion rates of V cholerae ranged from 2.4 X 107 to 1.1 X 109 cfu/g of stool. Of the V. cholerae colonies recovered from stool, >99.8% had a rugose morphology. Stool volumes for our volunteers did not differ significantly from those of past volunteers challenged with 106 cfu of a smooth N16961 variant; however, stool volumes of persons receiving rugose strains were significantly different from those reported after challenge with ~ 104 cfu of smooth forms of the strain (P < .02, Wilcoxon rank sum test). Peak V. cholerae counts in stool after rugose challenge were comparable to those reported for volunteers after challenge with smooth N 16961 variants. With the exception of the response to the matrix material itself, immunologic responses were similar to those seen in volunteers receiving smooth V. cholerae variants. In the ELISPOT assays, cells secreting IgG and IgA antibodies directed against CT were identified in all 6 volunteers. Antibody-secreting cells directed against Inaba and Ogawa LPSs and the matrix material were demonstrable in 1-5 volunteers, depending on the assay. All volunteers showed a strong serum IgG anti-CT response (mean peak optical density = 2.6). They also demonstrated vibriocidal responses to the smooth Inaba and Ogawa test strains (reciprocal geometric mean peak antibody titers of 6303 and 1810, respectively). However, when the rugose challenge strain was used in the vibriocidal assay, typical responses were not seen, and when contents of test wells were plated out, viable organisms could be demonstrated at all serum dilutions. Discussion Rugose strains represent an interesting and still poorly understood morphologic variant of V. cholerae. Our data suggest 1368 Concise Communications that all V. cholerae can assume this form, with conditions present in alkaline peptone water (a standard enrichment broth medium for V cholerae) selecting for or promoting the shift to rugose morphology. Rugose strains appear to produce an exopolysaccharide that promotes cell aggregation. Reminiscent of the protective effect of biofilms [12], this aggregation may shield individual cells from killing by disinfectants, such as chlorine, or lysis by complement. Vibrios are an important component of marine biofilms, and it is possible that the exopolysaccharide produced by V cholerae plays a role in marine biofilm formation; this, in tum, may contribute to attachment of bacteria to marine organisms, such as plankton. In this context, rugosity may represent a normal biologic adaptation of a species (V cholerae) that originates from marine and estuarine environments. Analogies to rugosity can be found in a number of other bacterial species, including the expression of alginate by mucoid strains of Pseudomonas aeruginosa and expression of an adhesive exopolysaccharide by the marine genus Hyphomonas. In contrast to rugose, rough V. cholerae strains have a modified or 0 antigen-deficient LPS [1, 13, 14]. It has generally been accepted (although without overwhelming data) that rough strains have decreased virulence [14, 15], and there has been a tendency to assume that virulence was confined to smooth V. cholerae variants. In prior studies at the Center for Vaccine Development, great care has always been taken to select smooth colonies for use in preparation of inocula for cholera challenges. As a result, we felt comfortable in assuming that prior challenges, conducted with N 16961 under identical experimental conditions, were representative of response to the smooth form of the strain. While numbers in volunteer studies of this type are of necessity small, our data suggest that the clinical syndrome and host immunologic response elicited by smooth and rugose variants are directly comparable. Since vibriocidal activity is dependent in part on complement-mediated killing (to which rugose forms show resistance), it is not unexpected that vibriocidal results differed in assays that incorporated the rugose challenge strain. In his 1940 paper on the rugose morphology, White [2] states that "it is difficult to escape the conclusion that the rugose substance is a protective secretion with a role in assisting the survival of the race [V cholerae] in nature." This comment has particular relevance in light of the apparent ability of rugose forms to survive in the presence of chlorine, which forms one of the first-line defenses against cholera. Chlorination is an effective intervention in controlling cholera [16]. However, if rugose strains are present in a water system, chlorination may simply decrease the number of viable V. cholerae rather than totally eradicate the organism. The persistence of viable cholera organisms in this setting may, in turn, contribute to further JID 1996; 174 (December) spread of the infection. There are limited observations suggesting that rugose forms are present in samples from clinical and environmental sources [2]. However, we still know little, if anything, about the impact of rugose strains on survival of V. cholerae in the environment and the epidemiology of the disease in human populations. Further studies in these areas are warranted. Acknowledgment We thank Michael Tanner for his excellent technical assistance in the flow cytometric analyses. References 1. White PB. The rugose variant of vibrios. J Pathol Bacteriol 1938;46: 1-6. 2. White PB. The characteristic hapten and antigen of rugose races of cholera and El Tor vibrios, J Pathol Bacteriol 1940; 50: 160-4. 3. White PB. A note on the globular forms of Vibrio cholerae. J Gen Microbiol 1950;4:36-7. 4. Rice EW, Johnson CH, Clark RM, et al. Vibrio cholerae 01 can assume a "rugose" survival form that resists killing by chlorine, yet retains virulence. Tnt J Environ Health Res 1993;3:89-98. 5. Levine MM, Black RE, Clements ML, Nalin DR, Cisneros L, Finkelstein RA. Volunteer studies in development of vaccines against cholera and enterotoxigenic Escherichia coli; a review. Tn: Home T, Holmgren J, Merson MH, Mollby R, eds. Acute enteric infections in children. New prospects for treatment and prevention. Amsterdam: Elsevier/NorthHolland Biomedical Press, 1981:443-59. 6. Morris JG Jr, Takeda T, Tall BD, et al. Experimental non-O group I Vibrio cholerae gastroenteritis in humans. J Clin Invest 1990; 85:697 - 705. 7. Hall RH, Vial PA, Kaper JB, Mekalanos JJ, Levine MM. Morphological studies on fimbriae expressed by Vibrio cholerae 01. Microb Pathog 1988;4:257 -65. 8. American Public Health Association. Standard methods for the examination of water and wastewater, 17th ed. Washington, DC: APHA, 1989. 9. Johnson JA, Panigrahi P, Morris JG Jr. Non-Ol Vibrio cholerae NRT36S produces a polysaccharide capsule that determines colony morphology, serum resistance, and virulence in mice. Infect Tmmun 1992;60:684-9. 10. Morris JG Jr, Losonsky GE, Johnson JA, et al. Clinical and immunologic characteristics of Vibrio cholerae 0139 Bengal infection in North American volunteers. J Infect Dis 1995; 171:903-8. 11. Levine MM, Young CR, Black RE, Takeda Y, Finkelstein RA. Enzymelinked immunosorbent assay to measure antibodies to purified heatlabile enterotoxins from human and porcine strains of Escherichia coli and to cholera toxin; application in serodiagnosis and seroepidemiology. J Clin Microbiol 1985;21: 174-9. 12. Marshall KC. Biofilms: an overview of bacterial adhesion, activity, and control at surfaces. ASM News 1992;58:202-7. 13. Kaper JB, Morris JG Jr, Levine MM. Cholera. Clin Microbiol Rev 1995; 8:48-86. 14. Barna D. Laboratory diagnosis of cholera. In: Barna D, Burrows W, eds. Cholera. Philadelphia: WB Saunders, 1974:85-126. 15. Sack RB, Miller CEo Progressive changes of vibrio serotypes in germ-free mice infected with V. cholerae. J Bacterial 1969; 99:688-95. 16. Deb BC, Sircar BK, Sengupta PG, et al. Studies on interventions to prevent El Tor cholera transmission in urban slums. Bull WHO 1986;64: 127-131.