Microbial Pathogenesis 1992 ; 12 : 451-458
Enteropathogenicity of non-toxigenic Vibrio cholerae 01 for adult mice Munshi Moyenuddin,' Kaye Wachsmuth, 2 * Stephen H . Richardson 3 and Warren L . Cook' 'Laboratory of Biological Sciences, Georgia State University, Atlanta, Georgia 30303, 2Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, and 'Department of Microbiology, The Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157, U.S.A . (Received January 21, 1992 ; accepted in revised form February 1, 1992)
Moyenuddin, M . (Laboratory of Biological Sciences, Georgia State University, Atlanta, Georgia 30303, U .S .A .), K . Wachsmuth, S . H . Richardson and W. L. Cook . Enteropathogenicity of non-toxigenic Vibrio cholerae 01 for adult mice . Microbial Pathogenesis 1992 ; 12 : 451-458 . The enteropathogenic potential of 32 Vibrio cholerae 01 isolates that do not produce cholera toxin was examined in the orally inoculated, sealed adult mouse model . Live cultures (2x10 1° cfu/ml) of 7/16 clinical and 6/16 environmental isolates produced a positive intestinal fluid accumulation (FA) ratio that reached near maximum at approximately 5 h post-inoculation . Colony hybridization did not detect genes for cholera toxin, Escherichia co/i heat-labile and heat-stable toxins, or shiga-like toxins . FA activity did not correlate precisely with cytotoxic activities on Chinese hamster ovary (28/32 positive), Vero (29/32) or HeLa (25/32) cells . Certain clinical and environmental isolates of non-toxigenic V. cholerae 01 appear to be enteropathogenic for the mouse, providing evidence that they may have pathogenic potential for humans through an as yet undefined mechanism(s) . Key words: Vibrio cholerae ; non-toxigenic ; enteropathogenic; enterotoxigenic ; mouse model ; DNA probes .
Introduction Vibrio cholerae serogroup 01 causes a fatal diarrheal disease in humans, primarily by secreting cholera toxin (CT) .' In recent years, strains of the same serogroup that do not produce CT (non-CT) have been isolated from patients with both intestinal and .` The enteropathogenic potential environment extraintestinal infections and from the and virulence properties of these strains are not clearly known and as such their public health importance remains uncertain . There have been reports on the secretory response in animal models of three clinical and three environmental isolates of non-CT V. cho/erae 01, but results of these studies have been contradictory and inconclusive . Different studies demonstrated that live cultures of two environmental isolates from Brazil (strains 1074-78 and 1196-78) and "Author to whom correspondence should be addressed .
0882-4010/92/060451 +08 $03 .00/0
© 1992 Academic Press Limited
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M . Moyenuddin et al.
Table 1 Isolation source, biotype and serogroup of the non-CT V. cholerae 01 strains Biotype
Serogroup °
Clinical isolates 2741-80 Stool, Fla . 2496-85 Stool, Fla . 1029-84 Stool, Fla . 884-82 Stool, Fla . 2740-80 Stool, Fla . 123-83 Stool, La . 2540-87 Stool, La . 917-84 Stool, Ga . 2483-85 Stool, Calif . 468-83 Stool, Pa . 2584-87 Stool, Peru 2583-87 Stool, Peru 2462-88 Stool, Australia 1165-77 Gall-bladder, Ala . 1077-79 Leg-wound, La . 2452-88 Blood, Miss .
El Tor Atypical' El Tor Atypical El Tor El Tor El Tor El Tor El Tor El Tor El Tor El Tor El Tor Atypical Atypical El Tor
Inaba Inaba Inaba Inaba Inaba Ogawa Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa Inaba Inaba Inaba
Environmental isolates 2168-81 Water, Fla . 2171-81 Water, Fla . 2169-81 Water, Fla . 692-79 Water, La . 1062-80 Water, La . V69-77 Water, Md . 3223-74 Water, Guam X316 Water, Guam 2843-80 Sewage, Fla . 2496-86 Sewage, La . 2497-86 Sewage, La . 2523-87 Sewage, La . 1196-78 Sewage, Brazil 1074-78 Sewage, Brazil 2633-78 Sewage, Brazil 2634-78 Sewage, Brazil
Atypical Atypical El Tor Atypical El Tor Atypical Atypical Atypical El Tor El Tor El Tor El Tor Atypical Atypical Atypical Atypical
Ogawa Inaba Inaba Inaba Ogawa Inaba Inaba Inaba Inaba Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa
Toxigenic controls 569B Stool, India 2524-87 Stool, La .
Classical El Tor
Inaba Inaba
Strains'
Source
'Strains were selected on the basis of epidemiologic data, case reports and geographic isolation site . They were obtained from the Centers for Disease Control culture collection . 'Slide agglutination was done to verify the serogroup and serotype of each strain . CDC polyvalent (A,B,C) antiserum, Inaba and Ogawa antisera were used to identify serotypes. `Atypical : strain that contains some characteristics of both the Classical and El Tor biogroups .
one environmental (strain V69-77) and three clinical isolates (strains 1077-79, 116577, 2740-80) from the United States, caused fluid accumulation (FA) either in the infant mouse intestine or in rabbit ligated ileal loops . 6' However, some of those strains (1074-78, V69-77, 1165-77) did not cause FA in infant mouse intestine when examined in another study 9 and the Brazil strains (1074-78, 1196-78) failed to produce diarrhea in human volunteers . 10 The FA-producing capacity of the culture supernatants has also varied in those studies . 6-8 In this study, we examined all the strains studied previously by others and additional clinical and environmental isolates of non-CT V. cholerae 01 (Table 1) in a genetically defined and immunocompetent adult mouse model (SAM) 11 to evaluate their FA
Enteropathogenicity of non-toxigenic V. cholerae 01
453
potential . Presence of enterotoxin genes was examined by colony hybridization and the production of cytotoxins 12 was tested in Chinese hamster ovary (CHO), Vero and HeLa cell assays .
Results
Enteropathogenic activity A total of 53 mice in a control group which received 4% NaHC0 3 in sterile H I B (Heat Infusion Broth) containing streptomycin, showed a mean FA ratio ±standard deviation (SD) of 86 .516±11 .625 (n = 10) at 2 h, 82 .0±6 .002 (n =12) at 4 h, 81 .181 ±8 .625 (n= 11) at 6 h, 83 .634±10 .217 (n= 10) at 8 h and 82 .2±6 .50 (n= 10) at 10 h . The mean FA value (+SD) produced by the negative control mice between 4-6 h was 88 .8 . Based on these values, FA ratios >100 were considered positive . Strains that produced a positive FA response in mice were termed enteropathogenic . Mice that received toxigenic controls and enteropathogenic strains producing FA ratio of > 120, enteropathogenic strains producing FA ratio of 100-106 and non-enteropathogenic strains (FA 100 and non-enteropathogenic strains mean FA ratios 0 .05) .
Colony hybridization None of the study strains hybridized with CT and E . coli LT, ST, or SLT gene-probes in the colony hybridization assay .
Cytotoxic activity Culture supernatants of 14 clinical and 14 environmental isolates were cytotoxic to CHO cells, 14 clinical and 15 environmental isolates were cytotoxic to Vero cells and
45 4
M . Moyenuddin et al.
Table 2 FA values (>100) and cytotoxic activities produced by non-CT enteropathogenic V. cholerae 01 strains Cytotoxicity titer°
Strains
FA values'
Clinical isolates 1029-84 2584-87 2452-88 2483-85 1077-79 2462-88 1165-77
149 .86 140 .01 139 .86 131 .83 106 .48 103 .26 102 .35
Environmental isolates 692-79 141 .70 1196-78 135.48 2168-81 124.65 121 .30 1062-80 2843-80 102 .0 V69-77 100 .19
CHO cells
Vero cells
HeLa cells
400 800 200 12 800 1600 3200
1280 2560 2560 160 320 320 80
16 64 64 32 32 32 8
160
160 20 20 80 10 1280
8
3200 10 200 12800
8 8 8 64
'FA values represent mean fluid accumulation ratios in six mice for each strain . 'The titers are expressed as the reciprocal of the highest dilution that exhibits cytotoxicity in 50% or more cells .
11 clinical and 14 environmental isolates were cytotoxic to HeLa cells . No significant difference was noted in the production of cytotoxicity by enteropathogenic and nonenteropathogenic strains . A non-enteropathogenic strain (2496-85) demonstrated the highest cytotoxicity titers in the tissue culture assays, whereas an enteropathogenic strain (1196-78) was negative in both the CHO and HeLa cell assays (Table 2 and 3) . Discussion and conclusion The SAM model has been used to evaluate the FA activities of purified and crude enterotoxins and of the organisms that produce them ." Positive results with two widely used standard strains of V. cholerae 01 (569B and CA401) and a non-01 strain (2194c) that was positive in ligated rabbit ileal loops 8 indicated that SAM offers a highly reproducible model for the study of the live organisms that are enteropathogenic for mouse . FA response of viable V. cholerae strains was also observed by other workers 9,13 in their orally inoculated infant mouse model . We did not examine normal flora E. co/i or killed V. cholerae strains in SAM . However, a group of 53 mice was examined for their FA response to the cell resuspending buffer and none of these mice accumulated any fluid in their intestines . Sterile culture broth and phosphate-buffered saline (PBS) have been used as negative 6,9,1a,14 One study compared a control in animal model assays by several researchers . non-pathogenic E. coil and culture broth as negative control in suckling mice model and observed that FA values produced by them were not statistically different .' Besides this, in spite of using a high challenge dose (1 x10' 0 cfu/ml), a large number of the test strains (59%) were negative in our model . CT-producing V. cholerae 01 strains were included as positive controls, not only to justify that the technics for the SAM
Enteropathogenicity of non-toxigenic V . cholerae 01
455
Table 3
FA values (
Enteropathogenicity of non-toxigenic Vibrio cholerae 01 for adult mice Munshi Moyenuddin,' Kaye Wachsmuth, 2 * Stephen H . Richardson 3 and Warren L . Cook' 'Laboratory of Biological Sciences, Georgia State University, Atlanta, Georgia 30303, 2Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, and 'Department of Microbiology, The Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157, U.S.A . (Received January 21, 1992 ; accepted in revised form February 1, 1992)
Moyenuddin, M . (Laboratory of Biological Sciences, Georgia State University, Atlanta, Georgia 30303, U .S .A .), K . Wachsmuth, S . H . Richardson and W. L. Cook . Enteropathogenicity of non-toxigenic Vibrio cholerae 01 for adult mice . Microbial Pathogenesis 1992 ; 12 : 451-458 . The enteropathogenic potential of 32 Vibrio cholerae 01 isolates that do not produce cholera toxin was examined in the orally inoculated, sealed adult mouse model . Live cultures (2x10 1° cfu/ml) of 7/16 clinical and 6/16 environmental isolates produced a positive intestinal fluid accumulation (FA) ratio that reached near maximum at approximately 5 h post-inoculation . Colony hybridization did not detect genes for cholera toxin, Escherichia co/i heat-labile and heat-stable toxins, or shiga-like toxins . FA activity did not correlate precisely with cytotoxic activities on Chinese hamster ovary (28/32 positive), Vero (29/32) or HeLa (25/32) cells . Certain clinical and environmental isolates of non-toxigenic V. cholerae 01 appear to be enteropathogenic for the mouse, providing evidence that they may have pathogenic potential for humans through an as yet undefined mechanism(s) . Key words: Vibrio cholerae ; non-toxigenic ; enteropathogenic; enterotoxigenic ; mouse model ; DNA probes .
Introduction Vibrio cholerae serogroup 01 causes a fatal diarrheal disease in humans, primarily by secreting cholera toxin (CT) .' In recent years, strains of the same serogroup that do not produce CT (non-CT) have been isolated from patients with both intestinal and .` The enteropathogenic potential environment extraintestinal infections and from the and virulence properties of these strains are not clearly known and as such their public health importance remains uncertain . There have been reports on the secretory response in animal models of three clinical and three environmental isolates of non-CT V. cho/erae 01, but results of these studies have been contradictory and inconclusive . Different studies demonstrated that live cultures of two environmental isolates from Brazil (strains 1074-78 and 1196-78) and "Author to whom correspondence should be addressed .
0882-4010/92/060451 +08 $03 .00/0
© 1992 Academic Press Limited
452
M . Moyenuddin et al.
Table 1 Isolation source, biotype and serogroup of the non-CT V. cholerae 01 strains Biotype
Serogroup °
Clinical isolates 2741-80 Stool, Fla . 2496-85 Stool, Fla . 1029-84 Stool, Fla . 884-82 Stool, Fla . 2740-80 Stool, Fla . 123-83 Stool, La . 2540-87 Stool, La . 917-84 Stool, Ga . 2483-85 Stool, Calif . 468-83 Stool, Pa . 2584-87 Stool, Peru 2583-87 Stool, Peru 2462-88 Stool, Australia 1165-77 Gall-bladder, Ala . 1077-79 Leg-wound, La . 2452-88 Blood, Miss .
El Tor Atypical' El Tor Atypical El Tor El Tor El Tor El Tor El Tor El Tor El Tor El Tor El Tor Atypical Atypical El Tor
Inaba Inaba Inaba Inaba Inaba Ogawa Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa Inaba Inaba Inaba
Environmental isolates 2168-81 Water, Fla . 2171-81 Water, Fla . 2169-81 Water, Fla . 692-79 Water, La . 1062-80 Water, La . V69-77 Water, Md . 3223-74 Water, Guam X316 Water, Guam 2843-80 Sewage, Fla . 2496-86 Sewage, La . 2497-86 Sewage, La . 2523-87 Sewage, La . 1196-78 Sewage, Brazil 1074-78 Sewage, Brazil 2633-78 Sewage, Brazil 2634-78 Sewage, Brazil
Atypical Atypical El Tor Atypical El Tor Atypical Atypical Atypical El Tor El Tor El Tor El Tor Atypical Atypical Atypical Atypical
Ogawa Inaba Inaba Inaba Ogawa Inaba Inaba Inaba Inaba Inaba Inaba Inaba Ogawa Ogawa Ogawa Ogawa
Toxigenic controls 569B Stool, India 2524-87 Stool, La .
Classical El Tor
Inaba Inaba
Strains'
Source
'Strains were selected on the basis of epidemiologic data, case reports and geographic isolation site . They were obtained from the Centers for Disease Control culture collection . 'Slide agglutination was done to verify the serogroup and serotype of each strain . CDC polyvalent (A,B,C) antiserum, Inaba and Ogawa antisera were used to identify serotypes. `Atypical : strain that contains some characteristics of both the Classical and El Tor biogroups .
one environmental (strain V69-77) and three clinical isolates (strains 1077-79, 116577, 2740-80) from the United States, caused fluid accumulation (FA) either in the infant mouse intestine or in rabbit ligated ileal loops . 6' However, some of those strains (1074-78, V69-77, 1165-77) did not cause FA in infant mouse intestine when examined in another study 9 and the Brazil strains (1074-78, 1196-78) failed to produce diarrhea in human volunteers . 10 The FA-producing capacity of the culture supernatants has also varied in those studies . 6-8 In this study, we examined all the strains studied previously by others and additional clinical and environmental isolates of non-CT V. cholerae 01 (Table 1) in a genetically defined and immunocompetent adult mouse model (SAM) 11 to evaluate their FA
Enteropathogenicity of non-toxigenic V. cholerae 01
453
potential . Presence of enterotoxin genes was examined by colony hybridization and the production of cytotoxins 12 was tested in Chinese hamster ovary (CHO), Vero and HeLa cell assays .
Results
Enteropathogenic activity A total of 53 mice in a control group which received 4% NaHC0 3 in sterile H I B (Heat Infusion Broth) containing streptomycin, showed a mean FA ratio ±standard deviation (SD) of 86 .516±11 .625 (n = 10) at 2 h, 82 .0±6 .002 (n =12) at 4 h, 81 .181 ±8 .625 (n= 11) at 6 h, 83 .634±10 .217 (n= 10) at 8 h and 82 .2±6 .50 (n= 10) at 10 h . The mean FA value (+SD) produced by the negative control mice between 4-6 h was 88 .8 . Based on these values, FA ratios >100 were considered positive . Strains that produced a positive FA response in mice were termed enteropathogenic . Mice that received toxigenic controls and enteropathogenic strains producing FA ratio of > 120, enteropathogenic strains producing FA ratio of 100-106 and non-enteropathogenic strains (FA 100 and non-enteropathogenic strains mean FA ratios 0 .05) .
Colony hybridization None of the study strains hybridized with CT and E . coli LT, ST, or SLT gene-probes in the colony hybridization assay .
Cytotoxic activity Culture supernatants of 14 clinical and 14 environmental isolates were cytotoxic to CHO cells, 14 clinical and 15 environmental isolates were cytotoxic to Vero cells and
45 4
M . Moyenuddin et al.
Table 2 FA values (>100) and cytotoxic activities produced by non-CT enteropathogenic V. cholerae 01 strains Cytotoxicity titer°
Strains
FA values'
Clinical isolates 1029-84 2584-87 2452-88 2483-85 1077-79 2462-88 1165-77
149 .86 140 .01 139 .86 131 .83 106 .48 103 .26 102 .35
Environmental isolates 692-79 141 .70 1196-78 135.48 2168-81 124.65 121 .30 1062-80 2843-80 102 .0 V69-77 100 .19
CHO cells
Vero cells
HeLa cells
400 800 200 12 800 1600 3200
1280 2560 2560 160 320 320 80
16 64 64 32 32 32 8
160
160 20 20 80 10 1280
8
3200 10 200 12800
8 8 8 64
'FA values represent mean fluid accumulation ratios in six mice for each strain . 'The titers are expressed as the reciprocal of the highest dilution that exhibits cytotoxicity in 50% or more cells .
11 clinical and 14 environmental isolates were cytotoxic to HeLa cells . No significant difference was noted in the production of cytotoxicity by enteropathogenic and nonenteropathogenic strains . A non-enteropathogenic strain (2496-85) demonstrated the highest cytotoxicity titers in the tissue culture assays, whereas an enteropathogenic strain (1196-78) was negative in both the CHO and HeLa cell assays (Table 2 and 3) . Discussion and conclusion The SAM model has been used to evaluate the FA activities of purified and crude enterotoxins and of the organisms that produce them ." Positive results with two widely used standard strains of V. cholerae 01 (569B and CA401) and a non-01 strain (2194c) that was positive in ligated rabbit ileal loops 8 indicated that SAM offers a highly reproducible model for the study of the live organisms that are enteropathogenic for mouse . FA response of viable V. cholerae strains was also observed by other workers 9,13 in their orally inoculated infant mouse model . We did not examine normal flora E. co/i or killed V. cholerae strains in SAM . However, a group of 53 mice was examined for their FA response to the cell resuspending buffer and none of these mice accumulated any fluid in their intestines . Sterile culture broth and phosphate-buffered saline (PBS) have been used as negative 6,9,1a,14 One study compared a control in animal model assays by several researchers . non-pathogenic E. coil and culture broth as negative control in suckling mice model and observed that FA values produced by them were not statistically different .' Besides this, in spite of using a high challenge dose (1 x10' 0 cfu/ml), a large number of the test strains (59%) were negative in our model . CT-producing V. cholerae 01 strains were included as positive controls, not only to justify that the technics for the SAM
Enteropathogenicity of non-toxigenic V . cholerae 01
455
Table 3
FA values (
