Although mixed bacterial infections of the gastrointestinal tract are common, comparisons of the clinical and immunological responses of patients coinfected and singly infected with major bacterial pathogens have been reported infrequently. In this study, we have focused on infections of hospitalized patients coinfected with the two most common bacterial diarrheal pathogens in Bangladesh, ETEC and V. cholerae O1. We show that mixed infection with the two pathogens is associated with more severe dehydration and vomiting and more intravenous fluid intake than what is seen for patients with ETEC diarrhea alone.
Studies of experimental animals have shown that dual infections of rotavirus and ETEC can cause more fluid loss and more prolonged diarrhea than infection with either pathogen alone (23
). Children infected with rotavirus together with other bacterial enteropathogens have more severe diarrhea and dehydration than children infected with rotavirus alone (15
). In the present study, although dual infection with ETEC and V. cholerae
O1 was more severe than infection with ETEC alone, there was no statistically significant difference in the severities of illness between dually infected patients and those with V. cholerae
O1 infection alone. Between 24 and 68% of our patients had taken antibiotics prior to hospitalization. The clinical severity of diarrhea upon presentation was not affected by whether or not they had taken antibiotics prior to hospitalization. Although information on the specific antibiotic taken was generally not available, our previous data indicated that the antibiotics taken by patients prior to hospitalization are usually not those recommended for either ETEC or V. cholerae
infection (our unpublished data).
We also observed that patients with ETEC alone and those with mixed infection sought care at the hospital after a comparatively longer duration of diarrhea than the cholera group. It is possible that for patients with mixed infection, ETEC was the primary pathogen, causing less severe initial diarrhea, followed by coinfection with V. cholerae O1 at a later point. This sequence might explain the longer duration of diarrhea at home in the VCET group than in the VC group.
The main finding of the study is that dual infection with both V. cholerae
O1 and ETEC produced significantly higher immune responses to cholera antigens than did infection with V. cholerae
O1 only. There are a number of possible explanations for this. Both CT and LT are powerful mucosal adjuvants (7
). Thus, the presence of the enterotoxins in dual infections may have produced an immunoadjuvant effect for responses to cholera antigens following infection. Clinical studies with oral cholera vaccines containing the cholera toxin B subunit have found these vaccines not only to give protection against cholera but also to augment protection against other enteric infections as well (13
). In Bangladesh, oral cholera vaccine protected against both LT-ETEC and LT/ST-ETEC diarrhea (7
). In travelers, the vaccine was found to have a protective effect against mixed infections with LT-ETEC and Salmonella
and also reduced ST-ETEC diarrhea (28
). Similarly, a CTB-based ETEC vaccine also showed protection against C. jejuni
and Salmonella enterica
serovar Typhi infections (14
). Derivatives of CT and LT have been explored as adjuvants in the development of Helicobacter pylori
). Previous studies have also shown that immune responses to a bivalent Salmonella
vaccine expressing the ETEC colonization factor CFA/I were enhanced by the coadministration of a mutant LT that was used as a mucosal adjuvant (16
In addition to acting as an immunoadjuvant (18
), recent data suggested that LT provides an advantage in the ability of bacteria to colonize epithelial cells in the small intestine. In addition, LT was shown to increase colonization by ETEC in both mouse and pig experimental models (1
). Adherence may be enhanced by the ADP-ribosylating activity of LT and by the levels of cyclic AMP (cAMP) that can affect host cell physiology (4
), leading to an enhanced adherence of V. cholerae
to the gut (20
However, we also observed increased immune responses in patients dually infected with V. cholerae
O1 and ST-expressing ETEC compared to responses in patients infected with V. cholerae
O1 alone. The heat-stable toxin (ST) is less well characterized for its immunomodulating properties than are LT and CT. Dual infections of children in developing countries with ETEC and enteropathogenic E. coli
(EPEC) have been observed (10
), and recent studies have shown that purified ST or crude extracts of ETEC can enhance the virulence of EPEC by use of laboratory-based in vitro
assay systems (8
). This suggested a molecular basis for the role of ST in causing an increased severity of disease in EPEC diarrhea. By analogy, ST might also have played a role in mediating the enhanced immunological responses in the VCET patients infected with ST-ETEC in the present study compared to those infected with V. cholerae
It is possible that both the adjuvant activity of LT and the effect of host-derived cAMP produced by either LT or ST to increase the adherence of V. cholerae
may have contributed to the enhanced immune responses seen in the dually infected patients compared to those infected with V. cholerae
alone in the present study. The increased CTB responses seen for dually infected patients may also reflect the antigenic cross-reactivity seen between LT and CT (6
In this study, we were not able to determine if the converse phenomenon is also true, i.e., if Vibrio cholerae O1 coinfection leads to enhanced immune responses to ETEC antigens as well. Since ETEC strains belong to a large number of different O and H groups, it was not possible to study the immune responses to these various antigens in this study with sufficient power to compare dually infected patients to those with ETEC infection alone. In addition, only 4 of the 25 patients with concomitant ETEC and V. cholerae O1 infection were positive for any of the 13 CFs that were tested in this study, and therefore, the responses to CFs were not determined either.
For preventive strategies against the major bacterial pathogens causing diarrhea, protective antitoxin immunity is an important target for vaccine development. Generally, for toxin-mediated enteric infections, robust responses are needed to overcome the effect of the toxins, for achieving protective efficacy for these major pathogens, and to formulate a wide-range global vaccine (8
). Since the presence of O-blood-group antigen was previously found to be associated with susceptibility to cholera (18
) and to be associated with the magnitude of immune responses generated after vaccination (21
), we analyzed the effect of this antigen on the immune responses seen for our three study groups. However, with the sizes of our study groups, we did not see a significant effect of the O blood group on the immune responses in the patients. Also, the proportions of patients of different blood groups were comparable across our study groups, suggesting the this was not the explanation for the observed increased immune responses in the VCET group compared to the immune responses of the VC group.
There was a significant difference in the proportions of ETEC strains that produced colonization factors between the patients in the VCET group and those in the ET group. We do not have an explanation for this difference at present, but we did not see a difference in immune responses between individuals in the VCET group infected with CF-positive strains and those infected with CF-negative ETEC strains.
Previous studies may suggest additional possible mechanisms for the increased immune responses seen in the dually infected group in the present study. Peltola et al. (28
) studied protective efficacy following vaccination with an oral cholera vaccine, an inactivated whole cell cholera toxin B subunit vaccine. In that study, in addition to the protective efficacy of this vaccine against ETEC diarrhea, there was also an unexpected protection against the combination of ETEC infection and infection with another pathogen, particularly mixed infections with Salmonella
. That study suggested that toxin may have a broader role in facilitating other enteric infections.
Glenn et al. (13
) recently suggested that LT and CT render the human gut more susceptible to colonization by enteric pathogens by disrupting innate mucosal defenses. Two possible mechanisms were suggested for this: toxin-mediated fluid secretion, disrupting the protective mucus layer, or toxin-mediated interference with the synthesis of antimicrobial peptides or other innate mucosal defenses. Those investigators suggested that the preconditioning of the gut by LT or CT enterotoxins might increase the efficiency of subsequent colonization by ETEC or other enteric pathogens. Thus, there may be advantages for V. cholerae
O1 and ETEC as coinfecting pathogens in terms of an enhanced capacity for colonization of the gut. This hypothesis is consistent with the observations here of an increased immune response to cholera antigens in dually infected patients compared to the responses of patients with single infection with V. cholerae
Both ETEC and V. cholerae O1 cause disease that gives protection from further infection and are thus considered to be vaccine preventable. Since vaccines are needed to protect individuals from these pathogens, a better understanding of the mechanisms leading to increased immune responses to dual infection with ETEC and V. cholerae O1 would be helpful in formulating appropriate and protective vaccines. Further studies of dual infection with ETEC and V. cholerae O1 and other bacterial pathogens in countries where these infections are endemic will shed additional light on these issues.